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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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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 ( av CV% = 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

We present a plasmonic biosensor capable of detecting the presence of bisphenol A in ultra-low concentrations, yielding a wavelength shift of 0.15 ± 0.01 nm in response to a solution of 1 fM concentration with limit of detection of 330 ± 70 aM The biosensing device consists of an array of gold nano-antennae with a total length of 2.3 cm that generate coupled localised surface plasmons (cLSPs) and is covalently modified with an aptamer specific for bisphenol A recognition. The array of nano-antennae is fabricated on a lapped section of standard telecommunication optical fibre, allowing for potential multiplexing and its use in remote sensing applications. These results have been achieved without the use of enhancement techniques and therefore the approach allows the direct detection of bisphenol A, a low molecular weight (228 Da) target usually detectable only by indirect detection strategies. Its detection at such levels is a significant step forward in measuring small molecules at ultra-low concentrations. Furthermore, this new sensing platform paves the way for the development of portable systems for in-situ agricultural measurements capable of retrieving data on a substance of very high concern at ultra-low concentrations., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

A Surface Plasmon Resonance imaging (SPRi) based DNA sensors for the selective and ultrasensitive human genomic DNA detection, directly extracted from lymphocytes (bypassing PCR amplification), is reported. To achieve DNA detection, a rationally chosen star-shaped nanoparticle (NP), namely gold nanostar (AuNS), has been applied, for the first time, in a sandwich-like assay based on the selective capturing of specific DNA targets and the subsequent signal amplification by a secondary DNA probe linked to AuNS. The plasmonic profile, size and electric field enhancements at the star tips contributed to the maximization of plasmon coupling between LSPs and SPs as aimed for analytical signal magnification. The system was first tested using short synthetic DNA target sequences and applied to DNA biosensing, lowering 610-fold the detection limit from 6.1 nM (without NSs labeling) to 10 pM (with NSs labeling). Then the biosensor was applied to genomic DNA samples, extracted from human lymphocytes and undergoing only to a simple ultrasonic fragmentation, lowering (~435 fold) the detection limit from 3.0 fM (without NSs labeling) to 6.9 aM (with NSs labeling). Thanks to the assay optimization, we proved that tuning the NSs surface coverage with DNA linked to nanoparticles is crucial not only for the increase of signals but also for the regenerability/reusability of the biosensor for tens of measurement cycles., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The mapping of specific single nucleotide polymorphisms (SNPs) in patients' genome is a main goal in theranostics, aiming to the development of therapies based on personalized medicine. In this review, Surface Plasmon Resonance (SPR) and Surface Plasmon Resonance imaging (SPRi) biosensors applied to the recognition of SNPs were reviewed, since these technologies are emerging in clinical diagnosis as powerful tools thanks to their analytical features, mainly the real-time and label-free monitoring based on array format for parallel analysis. Since the literature is heterogeneous, a critical classification and a systemic comparison of the analytical performances of published methods were here reviewed on the basis of the analytical strategy and the assay design. In particular, the use of helping agents (i.e. proteins, nanoparticles (NPs), intercalating agents) or artificial DNAs, often coupled to SPR to achieve allele discrimination and/or enhanced sensitivity, were here revised and classified. Finally, the real suitability of SPR biosensors to clinical diagnostics for SNPs detection was addressed by comparing their features and performances with those of other biosensors based on other techniques (e.g. electrochemical biosensors)., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Surface plasmon resonance imaging (SPRi) is at the forefront of optical sensing, allowing real-time and label free simultaneous multi-analyte measurements. It represents an interesting technology for studying a broad variety of affinity interactions with impact in chemistry, both in fundamental and applied research. Signal sampling and management is a key step in SPRi measurements to achieve successful performances. This work aims to develop a strategy for selecting the sensing areas, called Regions of Interest (ROIs), to be sampled for recording SPRi signals that could results in improved sensor performances. The approach has been evaluated using antigen-antibody interaction: anti-human IgGs are immobilized on the chip surface in an array format, while the specific ligand (hIgG antigen) is in solution. This approach has general applicability and demonstrates that rational selection of sensitive areas and standard management of SPRi data has dramatic impact on sensor behaviour. The criteria of the method are: (a) creation of high density maps of ROIs, (b) evaluation of the SPRi binding signals on all the ROIs during a pre-analysis step, (c) 3D elaboration of the results, and (d) ranking of the ROIs for their final selection in further biosensor analysis. Using standard solution of antigen, three different ROIs selection approaches have been compared for their analytical performances. The proposed innovative method results to be the best one for SPRi-based sensing applications., (Copyright © 2010 Elsevier B.V. All rights reserved.)

SPR imaging (SPRi) is at the forefront of optical label-free and real-time detection. It offers the possibility of monitoring hundreds of biological interactions simultaneously and from the binding profiles, allows the estimation of the kinetic parameters of the interactions between the immobilised probes and the ligands in solution. We review the current state of development of SPRi technology and its application including commercially available SPRi instruments. Attention is also given to surface chemistries for biochip functionalisation and suitable approaches to improve sensitivity., (Copyright 2009 Elsevier B.V. All rights reserved.)

Piezoelectric sensing is here applied to point mutation detection in human DNA. The mutation investigated is in the TP53 gene, which results inactivated in most cancer types. TP53 gene maps on chromosome 17 (17p13.1). It contains 11 exons and codifies for the relative protein, involved in cell proliferation. The TP53 gene has a wide mutation spectrum that is related to different tumours. In particular, those occurring in the structurally important L2 and L3 zinc-binding domains, have been linked to patient prognosis and more strongly to radiotherapy and chemotherapy resistance in several major cancers. For this reason, the identification of these mutations represents an important clinical target and biosensors could represent good candidate for fast mutation screening. In this paper, a DNA-based piezoelectric biosensor for the detection of the TP53 gene mutation at codon 248 is reported. A biotinylated probe was immobilised on the sensor surface via dextran-streptavidin modified surfaces. The sensor was optimised using synthetic oligonucleotides. Finally, the sensor system was successfully applied to polymerase chain reaction (PCR)-amplified real samples of DNA extracted from two cell lines, one normal (wild-type) and one mutated, carrying the mutation at codon 248 of the TP53 gene. The results obtained demonstrate that the DNA-based piezoelectric biosensor is able to detect the point mutations in PCR-amplified samples showing the potentialities of this approach for routine analysis.

A DNA-based surface plasmon resonance (SPR) biosensor has been developed for the detection of TP53 mutation using the inexpensive and commercially available instrument, SPREETA SPR-EVM-BT, from Texas Instruments. A direct immobilisation procedure, based on the coupling of thiol-derivatised oligonucleotide probes (Probe-C6-SH) to bare gold sensor surfaces, was optimized using synthetic oligonucleotides. Hybridisation reactions between the immobilised probe and a short sequence (26 mer) complementary, non-complementary and one-point mutation DNA were then investigated. The main analytical parameters of the sensor system were studied in detail including selectivity, sensitivity, reproducibility and analysis time. Finally, the sensor system was successfully applied to polymerase chain reaction (PCR)-amplified real samples, DNA extracted from both normal, wild-type, (Jurkat) and mutated (Molt 4), carrying the mutation at codon 248 of the TP53 cell lines. The results obtained demonstrate that the DNA-based SPR biosensor was able to distinguish sequences present in the various samples that differ only by one base; and hence, it appears to be a strong candidate technique for the detection of gene mutation.

An immobilisation procedure based on the direct coupling of thiol-derivatised oligonucleotide probes to bare gold sensor surfaces has been used for DNA sensing applications. The instrumentation used relies on surface plasmon resonance (SPR) transduction; in particular the commercially available instruments BIACORE X and SPREETA, have been employed in this study. The performances of the SPR-based DNA sensors resulting from direct coupling of thiol-derivatised DNA probes onto gold chips, have been studied in terms of the main analytical parameters, i.e. selectivity, sensitivity, reproducibility, analysis time, etc. A comparison between the thiol-derivatised immobilisation approach and a reference immobilisation method, based on the coupling of biotinylated oligonucleotide probes onto a streptavidin coated dextran sensor surface, using synthetic complementary oligonucleotides has been discussed. Finally, a denaturation method to obtain ssDNA ready for hybridisation analysis has been applied to polymerase chain reaction (PCR) amplified samples, for the detection of genetically modified organisms (GMOs).

In this paper, a simple and useful approach for DNA sensing based on surface plasmon resonance (SPR) transduction is reported. A new DNA sample pre-treatment has been optimised to allow fast and simple detection of hybridisation reaction between a target sequence in solution and a probe immobilised on the sensing surface. This pre-treatment consisted in a denaturation procedure of double stranded DNA containing the target sequence and was based on an high temperature treatment (95 degrees C, 5 min) followed by a 1 min incubation with small oligonucleotides. The oligonucleotides are designed to prevent the re-hybridising of the denatured strands, while enabling the target sequence to bind the immobilised probe. The important parameters of the procedure, i.e. incubation time, length and concentration of the oligonucleotides, have been studied in detail. The optimised DNA denaturation procedure has been successfully applied to the detection of amplified DNA with a commercially available SPR biosensor (Biacore X). DNA samples extracted from plant and human blood were tested after amplification by polymerase chain reaction (PCR).

The work evaluated a series of approaches to optimise detection of polymerase chain reaction (PCR) amplified DNA samples by an optical sensor based on surface plasmon resonance (SPR) (BiacoreX). The optimised procedure was based on an asymmetric PCR amplification system to amplify predominantly one DNA strand, containing the sequence complementary to a specific probe. The study moved into two directions, aiming to improve the analytical performance of SPR detection in PCR amplified products. One approach concerned the application of new strategies at the level of PCR, i.e. asymmetric PCR to obtain ssDNA amplified fragments containing the target capable of hybridisation with the immobilised complementary probe. The other strategy focused on the post-PCR amplification stage. Optimised denaturing conditions were applied to both symmetrically and asymmetrically amplified fragments. The effective combination of the two strategies allowed a rapid and specific hybridisation reaction. The developed method was successfully applied in the detection of genetically modified organisms.

A DNA piezoelectric sensor has been developed for the detection of genetically modified organisms (GMOs). Single stranded DNA (ssDNA) probes were immobilised on the sensor surface of a quartz crystal microbalance (QCM) device and the hybridisation between the immobilised probe and the target complementary sequence in solution was monitored. The probe sequences were internal to the sequence of the 35S promoter (P) and Nos terminator (T), which are inserted sequences in the genome of GMOs regulating the transgene expression. Two different probe immobilisation procedures were applied: (a) a thiol-dextran procedure and (b) a thiol-derivatised probe and blocking thiol procedure. The system has been optimised using synthetic oligonucleotides, which were then applied to samples of plasmidic and genomic DNA isolated from the pBI121 plasmid, certified reference materials (CRM), and real samples amplified by the polymerase chain reaction (PCR). The analytical parameters of the sensor have been investigated (sensitivity, reproducibility, lifetime etc.). The results obtained showed that both immobilisation procedures enabled sensitive and specific detection of GMOs, providing a useful tool for screening analysis in food samples.