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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.)

Optical genotyping of C3435T single nucleotide polymorphisms (SNPs) in unamplified human multidrug resistance (MDR1) gene was here performed by a surface plasmon resonance imaging (SPRi) dual-targeting DNA assay, allowing its selective detection down to 0.18 fM of the whole genomic DNA. The result was achieved by the combination of the rational selection of the DNA probe and an optimized sample pretreatment (i.e., ultrasound fragmentation and thermal denaturation). Some assay developments and tunings were reported in a previously published research, but here, for the first time, the biosensor reliability and its analytical performance were directly tested on the unamplified human DNA extracted from lymphocytes. The assay resulted to be able to differentiate among all the possible genotypes of C3435T (homozygote and heterozygote) in the diluted genomic samples using a label-free approach and by bypassing the classical PCR amplification of the target sequences. Moreover, the reusability of the DNA-based chip allowed up to 40 subsequent measuring cycles, opening new horizons in multi-SNP genotyping based on cheap and daily routine clinical monitoring by optical biosensing.

Nanoparticles with different sizes, refractive indices and plasmonic profiles are synthesized, labelled with DNA and embedded in DNA-based plasmonic nanoarchitectures. The contribution of the different properties to reflectivity variation % is rationally investigated by DNA hybridization measurements using the surface plasmon resonance imaging (SPRi) technology.

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.)

In the last 20 years, surface plasmon resonance (SPR) and its advancement with imaging (SPRi) emerged as a suitable and reliable platform in clinical analysis for label-free, sensitive, and real-time monitoring of biomolecular interactions. Thus, we report in this review the state of the art of clinical target detection with SPR-based biosensors in complex matrices (e.g., serum, saliva, blood, and urine) as well as in standard solution when innovative approaches or advanced instrumentations were employed for improved detection. The principles of SPR-based biosensors are summarized first, focusing on the physical properties of the transducer, on the assays design, on the immobilization chemistry, and on new trends for implementing system analytical performances (e.g., coupling with nanoparticles (NPs). Then we critically review the detection of analytes of interest in molecular diagnostics, such as hormones (relevant also for anti-doping control) and biomarkers of interest in inflammatory, cancer, and heart failure diseases. Antibody detection is reported in relation to immune disorder diagnostics. Subsequently, nucleic acid targets are considered for revealing genetic diseases (e.g., point mutation and single nucleotides polymorphism, SNPs) as well as new emerging clinical markers (microRNA) and for pathogen detection. Finally, examples of pathogen detection by immunosensing were also analyzed. A parallel comparison with the reference methods was duly made, indicating the progress brought about by SPR technologies in clinical routine analysis.

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.)

Biosensors represent an interesting tool in the search of bioactive compounds. In particular, optical sensors based on Surface Plasmon Resonance transduction (SPR) allow monitoring of biomolecular interaction in real time and without any labelling of the interactants. The biosensor analysis can be applied to both pure compounds or to complex mixtures (e.g. plant extract). The SPR detection principle is here presented and the application to the analysis of plant extracts (i.e. of Chelidonium majus L.) as a paradigmatic example for the search of bioactive compounds able to interact with DNA, is also discussed.

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.

Abnormal glycoconjugates have distinctly been recognized as potential biomarkers for cancer diagnosis. A great deal of attention has been focused on Tn antigen, an oversimplified mucin‐1 O‐glycan, over‐expressed in different cancers. Herein, we investigate the possibility to replace the use of anti‐Tn monoclonal antibodies with an innovative class of catecholamine‐based Molecularly Imprinted Polymers (MIPs), emerging in recent years as promising tools for bioanalytical applications. MIPs are synthetic receptors characterized by high sensitivity and specificity towards the imprinted target. Here, original polynorepinephrine‐based MIPs coupled to Surface Plasmon Resonance biosensing for Tn antigen recognition are reported. We have verified the imprinting and binding capacity of these MIPs towards very small antigenic entities, represented by the natural Tn antigen and the TnThr mimetic 1 (conjugated to BSA or linked to a MUC1 hexapeptide analogue), and compared the biosensor performances with an anti‐Tn monoclonal antibody. The results clearly display the effectiveness of the pursued imprinting strategies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Keywords: biomarker; molecularly imprinted polymers; polynorepinephrine; surface plasmon resonance; TACA; Tn antigen mimetic EN biomarker molecularly imprinted polymers polynorepinephrine surface plasmon resonance TACA Tn antigen mimetic 1 1 1 09/30/22 20220901 NES 220901 B The cover feature image shows b the possibility to replace the use of monoclonal antibodies with an innovative class of catecholamine-based Molecularly Imprinted Polymers (MIPs). Cover Feature: Structurally Constrained MUC1-Tn Mimetic Antigen as Template for Molecularly Imprinted Polymers (MIPs): A Promising Tool for Cancer Diagnostics (ChemPlusChem 9/2022) Biomarker, molecularly imprinted polymers, polynorepinephrine, surface plasmon resonance, TACA, Tn antigen mimetic. [Extracted from the article]

The use of nanoparticles (NPs) can substantially improve the analytical performance of surface plasmon resonance imaging (SPRi) in general, and in DNA sensing in particular. In this work, we report on the modification of the gold surface of commercial biochips with gold nanospheres, silica-coated gold nanoshells, and silver nanoprisms, respectively. The NPs were tethered onto the surface of the chip and functionalized with a DNA probe. The effects of tethering conditions and varying nanostructures on the SPRi signals were evaluated via hybridization assays. The results showed that coupling between planar surface plasmons and electric fields, generated by localized surface plasmons of the NPs, is mandatory for signal enhancement. Silver nanoprisms gave the best results in improving the signal change at a target DNA concentration of <50 nM by +50 % (compared to a conventional SPRi chip). The limit of detection for the target DNA was 0.5 nM which is 5 times less than in conventional SPRi. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: A composite monolayer consisting of a thiolipid and a nucleic acid probe (1), acts as a microenvironment for probing hybridization with the complementary strand (2) by means of impedance spectroscopy. The impedance measurements indicate a significant decrease in the resistance at the electrode surface upon the formation of the (1)/(2) duplex. The impedance measurements were performed in the absence of any amplifying label or added redox label. In order to characterize the electrodes surface and to follow the immobilization processes kinetics, surface plasmon resonance measurements were performed. [Copyright &y& Elsevier]

A biosensor approach using optical transduction based on surface plasmon resonance is adopted for the direct detection of target sequences in genomic DNA of several origins. In particular, the sensor was applied to three different sequences, representative of different living organisms from plant to human and differing in terms of copies in the relative genome. The sample pretreatment consisted in a fragmentation with restriction enzymes, followed by an optimized denaturation step. The biosensor resulted specific both with oligonucleotides and with genomic non-amplified DNA. The sensor could represent an alternative method to traditional biomolecular techniques for the identification of DNA sequences in digested DNA, bypassing the amplification step. [ABSTRACT FROM AUTHOR]

The development of a surface plasmon resonance (SPR) affinity biosensor based on DNA hybridisation is described. This biosensor has been applied to genetically modified organisms (GMOs) detection. Single stranded DNA (ssDNA) probes were immobilised on the sensor chip of an SPR device and the hybridisation between the immobilised probe and the complementary sequence (target) was monitored. The probe sequences were internal to the sequence of 35S promoter and NOS terminator which are inserted sequences in the genome of GMO regulating the transgene expression. The system has been optimised using synthetic oligonucleotides, then applied to real samples analysis. Samples, containing the transgenic target sequences, were amplified by polymerase chain reaction (PCR) and then detected with the SPR biosensor. [Copyright &y& Elsevier]

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. [Display omitted] [ABSTRACT FROM AUTHOR]

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. [Display omitted] [ABSTRACT FROM AUTHOR]

Immunotherapy has revolutionized modern medicine, becoming the largest part of the growing bio-drugs market. Along with the progress in this direction, the need for reliable monoclonal antibodies (mAbs) monitoring methods is a current foremost issue. In this scenario, the goal of this study was to design a straightforward beads-based plasmonic approach that combines magnetic beads (MBs) with a polynorepinephrine imprinted biopolymer (IBP) for real-time mAbs detection in biological matrices. Specifically, MBs-encoded by a specific antigen were exploited not only for the selective capture of the target mAb from human serum, but also MBs are directly involved in the molecular architecture of a sandwich assay. The mAb extraction from the real matrix occurred in a site-oriented manner by exploiting a paratope-epitope specific recognition, while leaving the constant mAb fragment (Fc) free to interact with an IBP specific for the mAb Fc portion. Anti-MYO-029, a recombinant monoclonal IgG1 antibody developed for myostatin inhibition and muscular-wasting disorders' treatment in clinical settings, was addressed as biological target to evaluate the analytical performances of the beads-based sensing assay. Myostatin-neutralizing antibodies may be misused as performance-enhancing drugs in sports competitions and their effective quantification has a central role also in the anti-doping control field. The optimization of the assay conditions led to establishing an assay able to achieve very good analytical performances in terms of repeatability and sensitivity, with negligible cross-reactivity with other Ig classes and IgG subclasses. In this frame, we proposed an extremely modulable assay, with potential large applicability, for multiplexing mAbs detection. • The detection of therapeutic monoclonal antibodies (mAb) used in immunotherapy is a current foremost issue. • An imprinted biopolymer against the constant mAb fragment (Fc) is coupled to magnetic beads encoded by specific antigens. • Beads-based affinity tool was directly involved in the molecular architecture of a sandwich plasmonic assay. • Anti-MYO-029 was addressed as biological target to evaluate the analytical performances of the beads-based sensing assay. [ABSTRACT FROM AUTHOR]

Early identification of neoplastic diseases is essential to achieve timely therapeutic interventions and significantly reduce the mortality of patients. A well-known biomarker is the Cancer Antigen 125 (CA125) or 16 mucin (MUC 16), a glycoprotein of the human family of mucins, already used for the diagnostic and prognostic evaluation of ovarian cancer. Therefore, the detection of CA125 to now remains a promising tool in the early diagnosis of this tumor. In this paper, we describe the development of RNA aptamers that bind with high affinity the tumor antigen CA125. We performed eight cycles of selection against CA125 purified protein. The selected aptamers were cloned and sequenced and the binding properties of the most promising sequences were studied by Real Time PCR and Surface Plasmon Resonance (SPR) to evaluate their ability in targeting CA125 protein with perspective applications in aptamer-based bioassays. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Surface plasmon resonance imaging (SPRi) was used as the transduction principle for the development of optical-based sensing for transgenes detection in human cell lines. The objective was to develop a multianalyte, label-free, and real-time approach for DNA sequences that are identified as markers of transgenosis events. The strategy exploits SPRi sensing to detect the transgenic event by targeting selected marker sequences, which are present on shuttle vector backbone used to carry out the transfection of human embryonic kidney (HEK) cell lines. Here, we identified DNA sequences belonging to the Cytomegalovirus promoter and the Enhanced Green Fluorescent Protein gene. System development is discussed in terms of probe efficiency and influence of secondary structures on biorecognition reaction on sensor; moreover, optimization of PCR samples pretreatment was carried out to allow hybridization on biosensor, together with an approach to increase SPRi signals by in situ mass enhancement. Real-time PCR was also employed as reference technique for marker sequences detection on human HEK cells. We can foresee that the developed system may have potential applications in the field of antidoping research focused on the so-called gene doping. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • A 'two-steps' competitive assay for gonadorelin, an emerging doping agent, is developed. • A polynorepinephrine-based MIP is synthesized for gonadorelin recognition in urine. • The MIP is prepared by simple coating of SPR biochips in few hours and mild conditions. • The MIP is capable to discriminate peptides differing for only a single amino acid. • A highly sensitive competitive bioassay is realized for anti-doping purposes. [ABSTRACT FROM AUTHOR]