Simona Scarano

Surface plasmon resonance imaging for affinity-based biosensors

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.

Surface plasmon resonance imaging (SPRi)-based sensing: A new approach in signal sampling and management

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.

Self-powered microneedle-based biosensors for pain-free high-accuracy measurement of glycaemia in interstitial fluid.

In this work a novel self-powered microneedle-based transdermal biosensor for pain-free high-accuracy real-time measurement of glycaemia in interstitial fluid (ISF) is reported. The proposed transdermal biosensor makes use of an array of silicon-dioxide hollow microneedles that are about one order of magnitude both smaller (borehole down to 4µm) and more densely-packed (up to 1×10(6)needles/cm(2)) than state-of-the-art microneedles used for biosensing so far. This allows self-powered (i.e. pump-free) uptake of ISF to be carried out with high efficacy and reliability in a few seconds (uptake rate up to 1µl/s) by exploiting capillarity in the microneedles. By coupling the microneedles operating under capillary-action with an enzymatic glucose biosensor integrated on the back-side of the needle-chip, glucose measurements are performed with high accuracy (±20% of the actual glucose level for 96% of measures) and reproducibility (coefficient of variation 8.56%) in real-time (30s) over the range 0-630mg/dl, thus significantly improving microneedle-based biosensor performance with respect to the state-of-the-art.

Affinity-based biosensors as promising tools for gene doping detection

Innovative bioanalytical approaches can be foreseen as interesting means for solving relevant emerging problems in anti-doping control. Sport authorities fear that the newer form of doping, so-called gene doping, based on a misuse of gene therapy, will be undetectable and thus much less preventable. The World Anti-Doping Agency has already asked scientists to assist in finding ways to prevent and detect this newest kind of doping. In this Opinion article we discuss the main aspects of gene doping, from the putative target analytes to suitable sampling strategies. Moreover, we discuss the potential application of affinity sensing in this field, which so far has been successfully applied to a variety of analytical problems, from clinical diagnostics to food and environmental analysis.

Direct detection of genomic DNA by surface plasmon resonance imaging: an optimized approach.

The direct detection of specific sequences in genomic DNA samples is very challenging in the biosensor-based approach. In this work we developed an optimized strategy for the direct detection of DNA sequences in human genomic samples by a surface plasmon resonance imaging technology. As model study, the target analyte was identified in a DNA sequence mapping the human ABCB1 gene. The computed-assisted approach was here applied for probe design. After a preliminary evaluation of the probe functioning by the complementary synthetic target, the system was applied to the direct detection of the target sequence in human genomic DNA extracted from lymphocytes. To achieve this result, several steps aimed to improve the analytical performances of the biosensor were studied and optimized. The immobilization chemistry, based on thiolated probes, was adapted here to non-amplified sequence detection. DNA sample pre-treatments, i.e. genomic fragmentation by ultrasounds and dsDNA denaturation by thermal treatment were also investigated. A sandwich-like strategy, by using a secondary probe, was also applied to understand and confirm the selectivity of the developed biosensor in detecting ABCB1 gene in genomic samples. Finally, a reliable calibration curve of ABCB1 was obtained with an experimental detection limit of 140 aM. Furthermore, the biosensor was well regenerable, assuring up to thirty cycles of effective measurements.

A reusable optical biosensor for the ultrasensitive and selective detection of unamplified human genomic DNA with gold nanostars.

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.

Simultaneous detection of transgenic DNA by surface plasmon resonance imaging with potential application to gene doping detection.

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.

Detecting Alzheimer's disease biomarkers: From antibodies to new bio-mimetic receptors and their application to established and emerging bioanalytical platforms - A critical review.

The failure of therapeutic treatment of Alzheimers disease (AD) patients can be related to the late onset of symptoms and, consequently, to a delayed pharmacological aid to counteract neurodegenerative progression. This is coupled to the fact that the diagnosis based on clinical criteria alone introduces high misdiagnosis rate. The availability of assessed biomarkers is therefore of crucial importance not only to counteract late diagnosis, but also to manage patients at high risk of AD development eligible for novel therapies. At the present time, amyloid-β peptides (Aβ1-40 and Aβ1-42 isoforms), alone or in combination with Tau protein (total and phosphorylated forms (p-tau)) constitute reliable AD biomarkers and result highly predictive of progression to AD dementia in patients with mild cognitive impairment (MCI), the earliest clinical presentation of AD. Improvement of existing diagnostic tools must take advantage of innovative bioanalytical approaches. In this review, starting from commercially available diagnostic platforms based on antibodies as recognition elements, we intended to provide a double point of view on the issue: 1) progresses achieved on innovative bioanalytical platforms (mainly sensors and biosensors) by using antibodies as consolidated receptors; 2) advance on promising bio-mimetic receptors alternative to antibodies in AD research, and their applications on conventional or innovative analytical platforms. In particular, we first focused on optical- (Propagating and Localized Surface Plasmon Resonance, named here SPR and LSPR) and electrochemical (voltammetric and impedimetric) transduction principles. Together with bioanalytical assays for AD biomarkers quantification, works aimed to investigate and understand their behavior, characteristics, and roles will also be considered in the discussion. An increasing interest in new emerging biomimetic receptors for AD diagnosis, as a promising alternative to antibodies is noticed, thus the description of peptides, peptoids, nanobodies, aptamers, and molecularly imprinted polymers and their role as recognition elements in different bioanalytical platforms is also reviewed. Features and limits are discussed, together with potentialities and perspectives of their further applicability to clinical routine AD analysis.

Single nucleotide polymorphism detection by optical DNA-based sensing coupled with whole genomic amplification

The work presented here deals with the optimization of a strategy for detection of single nucleotide polymorphisms based on surface plasmon resonance imaging. First, a sandwich-like assay was designed, and oligonucleotide sequences were computationally selected in order to study optimized conditions for the detection of the rs1045642 single nucleotide polymorphism in the gene ABCB1. Then the strategy was optimized on a surface plasmon resonance imaging biosensor using synthetic DNA sequences in order to evaluate the best conditions for the detection of a single mismatching base. Finally, the assay was tested on DNA extracted from human blood which was subsequently amplified using a whole genome amplification kit. The direct detection of the polymorphism was successfully achieved. The biochip was highly regenerable and reusable for up to 20 measurements. Furthermore, coupling these promising results with the multiarray assay, we can foresee applying this biosensor in clinical research extended to concurrent analysis of different polymorphisms.

Toward sensitive immuno-based detection of tau protein by surface plasmon resonance coupled to carbon nanostructures as signal amplifiers

Interest on Tau protein is fast increasing in Alzheimers disease (AD) diagnosis. There is the urgent need of highly sensitive and specific diagnostic platforms for its quantification, also in combination with the other AD hallmarks. Up to now, SPR has been poorly exploited for tau detection by immunosensing, due to sensitivity limits at nanomolar level, whereas the clinical requirement is in the picomolar range. Molecular architectures built in a layer-by-layer fashion, biomolecules and nanostructures (metallic or not) may amplify the SPR signal and improve the limit of detection to the desired sensitivity. Mostly gold nanostructures are widely employed to this aim, but great interest is also emerging in Multi Walled Carbon Nanotubes (MWCNTs). Here MWCNTs are modified and then decorated with the secondary antibody for tau protein. Eventually we took advantage from MWCNTs-antibody conjugate to obtain a sandwich-based bioassay with the capability to increase the SPR signal of about 102 folds compared to direct detection and conventional unconjugated sandwich. With respect to these results, we hope to give a strong impulse for further investigation on studying possible roles of carbon nanotubes in optical-based biosensing.