An α-helical peptide-based plasmonic biosensor for highly specific detection of α-synuclein toxic oligomers.

BACKGROUND: α-Synuclein (αS) aggregation is the main neurological hallmark of a group of neurodegenerative disorders, collectively referred to as synucleinopathies, of which Parkinson's disease (PD) is the most prevalent. αS oligomers are elevated in the cerebrospinal fluid (CSF) of PD patients, standing as a biomarker for disease diagnosis. However, methods for early PD detection are still lacking. We have recently identified the amphipathic 22-residue peptide PSMα3 as a high-affinity binder of αS toxic oligomers. PSMα3 displayed excellent selectivity and reproducibility, binding to αS toxic oligomers with affinities in the low nanomolar range and without detectable cross-reactivity with functional monomeric αS. RESULTS: In this work, we leveraged these PSMα3 unique properties to design a plasmonic-based biosensor for the direct detection of toxic oligomers under label-free conditions. SIGNIFICANCE AND NOVELTY: We describe the integration of the peptide in a lab-on-a-chip plasmonic platform suitable for point-of-care measurements of αS toxic oligomers in CSF samples in real-time and at an affordable cost, providing an innovative biosensor for PD early diagnosis in the clinic.

Validation of a plasmonic-based serology biosensor for veterinary diagnosis of COVID-19 in domestic animals.

The coronavirus disease 2019 (COVID-19) pandemic recently demonstrated the devastating impact on public health, economy, and social development of zoonotic infectious diseases, whereby viruses jump from animals to infect humans. Due to this potential of viruses to cross the species barrier, the surveillance of infectious pathogens circulation in domestic and close-to-human animals is indispensable, as they could be potential reservoirs. Optical biosensors, mainly those based on Surface Plasmon Resonance (SPR), have widely demonstrated its ability for providing direct, label-free, and quantitative bioanalysis with excellent sensitivity and reliability. This biosensor technology can provide a powerful tool to the veterinary field, potentially being helpful for the monitoring of the infection spread. We have implemented a multi-target COVID-19 serology plasmonic biosensor for the rapid testing and screening of common European domestic animals. The multi-target serological biosensor assay enables the detection of total SARS-CoV-2 antibodies (IgG + IgM) generated towards both S and N viral antigens. The analysis is performed in less than 15 min with a low-volume serum sample (<20 µL, 1:10 dilution), reaching a limit of detection of 49.6 ng mL-1. A complete validation has been carried out with hamster, dog, and cat sera samples (N = 75, including 37 COVID-19-positive and 38 negative samples). The biosensor exhibits an excellent diagnostic sensitivity (100 %) and good specificity (71.4 %) for future application in veterinary settings. Furthermore, the biosensor technology is integrated into a compact, portable, and user-friendly device, well-suited for point-of-care testing. This study positions our plasmonic biosensor as an alternative and reliable diagnostic tool for COVID-19 serology in animal samples, expanding the applicability of plasmonic technologies for decentralized analysis in veterinary healthcare and animal research.

Real-time monitoring of fenitrothion in water samples using a silicon nanophotonic biosensor.

Due to the large quantities of pesticides extensively used and their impact on the environment and human health, a prompt and reliable sensing technique could constitute an excellent tool for in-situ monitoring. With this aim, we have applied a highly sensitive photonic biosensor based on a bimodal waveguide interferometer (BiMW) for the rapid, label-free, and specific quantification of fenitrothion (FN) directly in tap water samples. After an optimization protocol, the biosensor achieved a limit of detection (LOD) of 0.29 ng mL-1 (1.05 nM) and a half-maximal inhibitory concentration (IC50) of 1.71 ng mL-1 (6.09 nM) using a competitive immunoassay and employing diluted tap water. Moreover, the biosensor was successfully employed to determine FN concentration in blind tap water samples obtaining excellent recovery percentages with a time-to-result of only 20 min without any sample pre-treatment. The features of the biosensor suggest its potential application for real time, fast and sensitive screening of FN in water samples as an analytical tool for the monitoring of the water quality.

One-Step Immobilization of Antibodies and DNA on Gold Sensor Surfaces via a Poly-Adenine Oligonucleotide Approach.

Label-free plasmonic biosensors have demonstrated promising capabilities as analytical tools for the detection of virtually any type of biomarker. They are presented as good candidates for precision diagnostics since they offer highly sensitive, cost-effective solutions that can be used in any clinical or laboratory setting without the need for specialized trainees. However, different surface functionalization protocols are required, depending on the nature of the biorecognition element, limiting their capabilities for integrated multi-biomarker detection. Here, we present a simple, yet efficient, one-step immobilization approach that is common for both DNA probes and antibodies. Our immobilization approach relies on the incorporation of poly-adenine (polyA) blocks in both nucleic acid probes and antibodies. PolyA sequences have a remarkable affinity for gold surfaces and can specifically interact with sufficient strength to generate stable, dense, and highly ordered monolayers. We have demonstrated excellent performance of our universal functionalization method, showing limits of detection and quantification in the pM-nM range. Moreover, it was able to reduce up to 50% of the background signal from undiluted serum samples compared to conventional methods, demonstrating the immense potential of this strategy for the direct analysis of human biofluids, essential for rapid point-of-care diagnostics. The polyA-based immobilization approach is a promising alternative for the generation of multiplexed biosensors that can detect both protein and nucleic acid biomarkers for multiparametric diagnostic assays.

A compact SPR biosensor device for the rapid and efficient monitoring of gluten-free diet directly in human urine.

Celiac disease (CD) is a chronic autoimmune disorder induced in genetically susceptible individuals by the ingestion of gluten from wheat, rye, barley, or certain varieties of oats. A careful diet follow-up is necessary to avoid health complications associated with long-term gluten intake by the celiac patients. Small peptides (GIP, gluten immunogenic peptides) derived from gluten digestion, which are excreted in the urine and feces, have emerged as promising biomarkers to monitor gluten intake. We have implemented a simple and sensitive label-free point-of-care (POC) device based on surface plasmon resonance for the direct detection of these biomarkers in urine. The assay employs specific monoclonal antibodies and has been optimized for the detection of the 33-mer α2-gliadin, known as the main immunogenic peptide of wheat gluten, and for the detection of GIP. Direct detection in undiluted urine has been accomplished by using biosensing chips containing a robust and stable biorecognition layer, obtained after carefully optimizing the biofunctionalization protocol. Excellent limits of detection have been reached (1.6-4.0 ng mL-1 using mAb G12 and A1, respectively), which ensures the detection of gluten peptides even when the gluten intake is around the maximum tolerable amount in the digestive tract (< 50 mg) for celiac individuals. No sample pretreatment, extraction, or dilution is required, and the analysis takes less than 15 min. The assays have excellent reproducibility' as demonstrated by measuring spiked urine samples containing the same target concentration using different biofunctionalized chips prepared and stored at different periods of time (i.e., CV% of 3.58% and 11.30%, for G12- and A1-based assays, respectively). The assay has been validated with real samples. These features pave the way towards an end-user easy-to-handle biosensor device for the rapid monitoring of gluten-free diet (GFD) and follow-up of the health status in celiac patients.

Optical nanogap antennas as plasmonic biosensors for the detection of miRNA biomarkers.

Nanoplasmonic biosensors based on nanogap antenna structures usually demand complex and expensive fabrication processes in order to achieve a good performance and sensitive detection. We here report the fabrication of large-area nanoplasmonic sensor chips based on nanogap antennas by employing a customized, simple and low-cost colloidal lithography process. By precisely controlling the angle for tilted e-beam metal evaporation, an elliptical mask is produced, which defines the total length of the dipole antenna nanostructures while assuring that the plasmonic response is oriented in the same direction along the sensor chip. Large-area sensor chips of nanogap antennas formed by pairs of gold nanodisks separated by gaps with an average size of 11.6 ± 4.7 nm are obtained. The optical characterization of the nanogap antenna structures in an attenuated total reflection (ATR) configuration shows a bulk refractive index sensitivity of 422 nm per RIU, which is in agreement with FDTD numerical simulations. The biosensing potential of the cm2-sized nanostructured plasmonic sensor chips has been evaluated for the detection of miRNA-210, a relevant biomarker for lung cancer diagnosis, through a DNA/miRNA hybridization assay. A limit of detection (LOD) of 0.78 nM (5.1 ng mL-1) was achieved with no need of further amplification steps, demonstrating the high sensitivity of these plasmonic nanogap antennas for the direct and label-free detection of low molecular weight biomolecules such as miRNAs.

Label-free detection of nosocomial bacteria using a nanophotonic interferometric biosensor.

Nosocomial infections are a major concern at the worldwide level. Early and accurate identification of nosocomial pathogens is crucial to provide timely and adequate treatment. A prompt response also prevents the progression of the infection to life-threatening conditions, such as septicemia or generalized bloodstream infection. We have implemented two highly sensitive methodologies using an ultrasensitive photonic biosensor based on a bimodal waveguide interferometer (BiMW) for the fast detection of Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA), two of the most prevalent bacteria associated with nosocomial infections. For that, we have developed a biofunctionalization strategy based on the use of a PEGylated silane (silane-PEG-COOH) which provides a highly resistant and bacteria-repelling surface, which is crucial to specifically detect each bacterium. Two different biosensor assays have been set under standard buffer conditions: one based on a specific direct immunoassay employing polyclonal antibodies for the detection of P. aeruginosa and another one employing aptamers for the direct detection of MRSA. The biosensor immunoassay for P. aeruginosa is fast (it only takes 12 min) and specific and has experimentally detected concentrations down to 800 cfu mL-1 (cfu: colony forming unit). The second one relies on the use of an aptamer that specifically detects penicillin-binding protein 2a (PBP2a), a protein only expressed in the MRSA mutant, providing a photonic biosensor with the ability to identify the resistant pathogen MRSA and differentiate it from methicillin-susceptible S. aureus (MSSA). Direct, label-free, and selective detection of whole MRSA bacteria has been achieved, making possible the direct detection of also 800 cfu mL-1. According to the signal-to-noise (S/N) ratio of the device, a theoretical limit of detection (LOD) of around 49 and 29 cfu mL-1 was estimated for P. aeruginosa and MRSA, respectively. Both results obtained under standard conditions reveal the great potential this interferometric biosensor device has as a versatile and specific tool for bacterial detection and quantification, providing a rapid method for the identification of nosocomial pathogens within the clinical requirements of sensitivity for the diagnosis of infections.

Early sepsis diagnosis via protein and miRNA biomarkers using a novel point-of-care photonic biosensor.

Sepsis is a condition characterized by a severe stage of blood-infection often leading to tissue damage, organ failure and finally death. Fast diagnosis and identification of the sepsis stage (sepsis, severe sepsis or septic shock) is critical for the patient's evolution and could help in defining the most adequate treatment in order to reduce its mortality. The combined detection of several biomarkers in a timely, specific and simultaneous way could ensure a more accurate diagnosis. We have designed a new optical point-of-care (POC) device based on a phase-sensitive interferometric biosensor with a label-free microarray configuration for potential high-throughput evaluation of specific sepsis biomarkers. The sensor chip, which relies on the use of metallic nanostructures, provides versatility in terms of biofunctionalization, allowing the efficient immobilization of different kind of receptors such as antibodies or oligonucleotides. We have focused on two structurally different types of biomarkers: proteins, including C-reactive protein (CRP) and Interleukin 6 (IL6), and miRNAs, using miRNA-16 as an example. Limits of Detection (LoD) of 18 µg mL-1, 88 µg mL-1 and 1 µM (6 µg mL-1) have been respectively obtained for CRP, IL6 and miRNA-16 in individual assays, with high accuracy and reproducibility. The multiplexing capabilities have also been assessed with the simultaneous analysis of both protein biomarkers.

Nanoplasmonic biosensor device for the monitoring of acenocoumarol therapeutic drug in plasma.

Acenocoumarol (Sintrom®) is an oral anticoagulant prescribed for the treatment of a variety of thromboembolic disorders such as atrial fibrillation and thrombosis or embolism. It inhibits fibrin production preventing clot formation. Acenocoumarol has a narrow therapeutic range, and its effects depend on several factors, such as body weight, age, metabolism, diet, certain medical conditions or the intake of additional drugs, among others. A higher dose may result in the risk of bleeding, while if it is too low, the risk of blood clot can increase. Complementary tools that allow the therapeutic drug monitoring (TDM) of acenocoumarol plasmatic levels from the starting of the treatment would be of paramount importance to personalize the treatment. Point-of-care (POC) devices can offer an added value in facilitating on-site monitoring (i.e. hospitals, primary care doctor or even by the patient itself) and can aid in dosage management. With this aim, we have developed a compact and simple nanoplasmonic sensing device based on gold nanodisks for the rapid monitoring of acenocoumarol, using highly specific polyclonal antibodies produced against this drug. A specific and reproducible label free indirect competitive assay has been developed and the viability of performing the evaluation directly in plasma diluted 1:1 has been demonstrated. A limit of detection (LOD) of only 0.77 ±â€¯0.69 nM, an IC50 of 48.2 ±â€¯5.12 nM and a dynamic range between 3.38 ±â€¯1.33 nM and 1154 ±â€¯437 nM were achieved, which easily fit within the drug plasma levels of acenocoumarol, making this approach a highly attractive option for its decentralized monitoring in human plasma.

Gold/silver/gold trilayer films on nanostructured polycarbonate substrates for direct and label-free nanoplasmonic biosensing.

Ultrasmooth gold/silver/gold trilayer nanostructured plasmonic sensors were obtained using commercial Blu-ray optical discs as nanoslits-based flexible polymer substrates. A thin gold film was used as an adhesion and nucleation layer to improve the chemical stability and reduce the surface roughness of the overlying silver film, without increasing ohmic plasmon losses. The structures were physically and optically characterized and compared with nanostructures of single gold layer. Ultrasmooth and chemically stable trilayer nanostructures with a surface roughness <0.5 nm were obtained following a simple and reproducible fabrication process. They showed a figure of merit (FOM) value up to 69.2 RIU-1 which is significantly higher (more than 95%) than the gold monolayer counterpart. Their potential for biosensing was demonstrated by employing the trilayer sensor for the direct and refractometric (label-free) detection of C-reactive protein (CRP) biomarker in undiluted urine achieving a Limit of Detection (LOD) in the pM order.

A label-free nanostructured plasmonic biosensor based on Blu-ray discs with integrated microfluidics for sensitive biodetection.

Nanostructure-based plasmonic biosensors have quickly positioned themselves as interesting candidates for the design of portable optical biosensor platforms considering the potential benefits they can offer in integration, miniaturization, multiplexing, and real-time label-free detection. We have developed a simple integrated nanoplasmonic sensor taking advantage of the periodic nanostructured array of commercial Blu-ray discs. Sensors with two gold film thicknesses (50 and 100nm) were fabricated and optically characterized by varying the oblique-angle of the incident light in optical reflectance measurements. Contrary to the use normal light incidence previously reported with other optical discs, we observed an enhancement in sensitivity and a narrowing of the resonant linewidths as the light incidence angle was increased, which could be related to the generation of Fano resonant modes. The new sensors achieve a figure of merit (FOM) up to 35 RIU-1 and a competitive bulk limit of detection (LOD) of 6.3×10-6 RIU. These values significantly improve previously reported results obtained with normal light incidence reflectance measurements using similar structures. The sensor has been combined with versatile, simple, ease to-fabricate microfluidics. The integrated chip is only 1cm2 (including a PDMS flow cell with a 50µm height microfluidic channel fabricated with double-sided adhesive tape) and all the optical components are mounted on a 10cm×10cm portable prototype, illustrating its facile miniaturization, integration and potential portability. Finally, to assess the label-free biosensing capability of the new sensor, we have evaluated the presence of specific antibodies against the GTF2b protein, a tumor-associate antigen (TAA) related to colorectal cancer. We have achieved a LOD in the pM order and have assessed the feasibility of directly measuring biological samples such as human serum.

Label-free nanoplasmonic sensing of tumor-associate autoantibodies for early diagnosis of colorectal cancer.

Colorectal cancer is treatable and curable when detected at early stages. However there is a lack of less invasive and more specific screening and diagnosis methods which would facilitate its prompt identification. Blood circulating autoantibodies which are immediately produced by the immune system at tumor appearance have become valuable biomarkers for preclinical diagnosis of cancer. In this work, we present the rapid and label-free detection of colorectal cancer autoantibodies directly in blood serum or plasma using a recently developed nanoplasmonic biosensor. Our nanoplasmonic device offers sensitive and real-time quantification of autoantibodies with excellent selectivity and reproducibility, achieving limits of detection around 1 nM (150-160 ng mL(-1)). A preliminary evaluation of clinical samples of colorectal cancer patients has shown good correlation with ELISA. These results demonstrate the reliability of the nanobiosensor strategy and pave the way towards the achievement of a sensitive diagnostic tool for early detection of colorectal cancer.

Label-free SPR detection of gluten peptides in urine for non-invasive celiac disease follow-up.

Motivated by the necessity of new and efficient methods for dietary gluten control of celiac patients, we have developed a simple and highly sensitive SPR biosensor for the detection of gluten peptides in urine. The sensing methodology enables rapid and label-free quantification of the gluten immunogenic peptides (GIP) by using G12 mAb. The overall performance of the biosensor has been in-depth optimized and evaluated in terms of sensitivity, selectivity and reproducibility, reaching a limit of detection of 0.33 ng mL(-1). Besides, the robustness and stability of the methodology permit the continuous use of the biosensor for more than 100 cycles with excellent repeatability. Special efforts have been focused on preventing and minimizing possible interferences coming from urine matrix enabling a direct analysis in this fluid without requiring extraction or purification procedures. Our SPR biosensor has proven to detect and identify gluten consumption by evaluating urine samples from healthy and celiac individuals with different dietary gluten conditions. This novel biosensor methodology represents a novel approach to quantify the digested gluten peptides in human urine with outstanding sensitivity in a rapid and non-invasive manner. Our technique should be considered as a promising opportunity to develop Point-of-Care (POC) devices for an efficient, simple and accurate gluten free diet (GFD) monitoring as well as therapy follow-up of celiac disease patients.

Design of a surface plasmon resonance immunoassay for therapeutic drug monitoring of amikacin.

The therapeutic drug monitoring (TDM) of pharmaceutical drugs with narrow therapeutic ranges is of great importance in the clinical setting. It provides useful information towards the enhancement of drug therapies, aiding in dosage control and toxicity risk management. Amikacin is an aminoglycoside antibiotic commonly used in neonatal therapies that is indicated for TDM due to the toxicity risks inherent in its use. Current techniques for TDM such as high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) are costly, time consuming, and cannot be performed at the site of action. Over the last decades, surface plasmon resonance (SPR) biosensors have become increasingly popular in clinical diagnostics due to their ability to detect biomolecular interactions in real-time. We present an SPR-based competitive immunoassay for the detection of the antibiotic amikacin, suitable for TDM in both adults and neonates. We have obtained high specificity and sensitivity levels with an IC50 value of 1.4ng/mL and a limit of detection of 0.13ng/mL, which comfortably comply with the drug's therapeutic range. Simple dilution of serum can therefore be sufficient to analyze low-volume real samples from neonates, increasing the potential of the methodology for TDM. Compared to current TDM conventional methods, this SPR-based immunoassay can provide advantages such as simplicity, potential portability, and label-free measurements with the possibility of high throughput. This work is the foundation towards the development of an integrated, simple use, highly sensitive, fast, and point-of-care sensing platform for the opportune TDM of antibiotics and other drugs in a clinical setting.

Highly sensitive dendrimer-based nanoplasmonic biosensor for drug allergy diagnosis.

A label-free biosensing strategy for amoxicillin (AX) allergy diagnosis based on the combination of novel dendrimer-based conjugates and a recently developed nanoplasmonic sensor technology is reported. Gold nanodisks were functionalized with a custom-designed thiol-ending-polyamido-based dendron (d-BAPAD) peripherally decorated with amoxicilloyl (AXO) groups (d-BAPAD-AXO) in order to detect specific IgE generated in patient's serum against this antibiotic during an allergy outbreak. This innovative strategy, which follows a simple one-step immobilization procedure, shows exceptional results in terms of sensitivity and robustness, leading to a highly-reproducible and long-term stable surface which allows achieving extremely low limits of detection. Moreover, the viability of this biosensor approach to analyze human biological samples has been demonstrated by directly analyzing and quantifying specific anti-AX antibodies in patient's serum without any sample pretreatment. An excellent limit of detection (LoD) of 0.6ng/mL (i.e. 0.25kU/L) has been achieved in the evaluation of clinical samples evidencing the potential of our nanoplasmonic biosensor as an advanced diagnostic tool to quickly identify allergic patients. The results have been compared and validated with a conventional clinical immunofluorescence assay (ImmunoCAP test), confirming an excellent correlation between both techniques. The combination of a novel compact nanoplasmonic platform and a dendrimer-based strategy provides a highly sensitive label free biosensor approach with over two times better detectability than conventional SPR. Both the biosensor device and the carrier structure hold great potential in clinical diagnosis for biomarker analysis in whole serum samples and other human biological samples.

Efectividad de una intervención enfermera en la mejora del estado nutricional de pacientes con trastornos de salud mental hospitalizados / Efficacy of nursing intervention in the improvement of the nutritional status of hospitalized patients with mental health disorders

OBJETIVO: evaluar la contribución de un programa fundamentado en el Proceso de Atención de Enfermería y dirigido a la mejora del estado nutricional de pacientes con trastorno mental hospitalizados. MÉTODO: estudio de intervención antes-después en pacientes ingresados en la unidad psiquiátrica de agudos, con malnutrición o riesgo, durante el primer cuatrimestre del año 2012. La variable dependiente principal fue el estado nutricional medido a través del Mini Nutritional Assessment (MNA); también fueron utilizados los indicadores para los objetivos NOC (Nursing Outcome Classification): apetito, estado nutricional, conducta de mantenimiento del peso, y autocuidados: comer. La intervención NIC (Nursing Intervention Classification) llevada a cabo fue «Manejo de la nutrición» para la mejora del estado nutricional. Para la comparación de las medias de las diferentes mediciones (MNA antes y después) se utilizó la t de Student para datos apareados. RESULTADOS: fueron estudiados 74 pacientes. Un 47,3% tenía trastornos del estado de ánimo, y el 43,2% esquizofrenia. Al alta de la unidad se encontraron diferencias estadísticamente significativas en todas las variables analizadas. La mejora del estado nutricional, según el cuestionario MNA, se produjo tanto en la escala A (diferencia de medias: -2,23; IC95%: -2,67, -1,79) como en la escala B (diferencia de medias: -1,54; IC 95%: -2,02, -1,05). CONCLUSIONES: la intervención de Enfermería NIC «Manejo de la nutrición», como eje del programa nutricional, ha contribuido a mejorar el estado nutricional de los pacientes hospitalizados, reflejado en la mejora de estado nutricional según el cuestionario MNA, en la disminución del grado de dependencia en alimentación y en la consecución de objetivos NOC relacionados

OBJECTIVE: to assess the potential contribution of a program based on the Nursing Care Process, targeted to the improvement of the nutritional status of hospitalized patients with mental disorders. METHOD: interventional before-after study, conducted on patients hospitalized in the Acute Psychiatric Unit, with malnutrition or at risk, during the first four months of 2012. The primary dependent variable was the nutritional status measured through the Mini Nutritional Assessment (MNA); the NOC (Nursing Outcome Classification) objective indicators were also used: appetite, nutritional status, weight maintenance behaviour, and self-care: eating. The NIC (Nursing Intervention Classification) intervention conducted was «Nutrition Management» for the improvement of nutritional status. For comparison of mean levels of the different measures (MNA before and after), Student's t test was used for paired data. RESULTS: seventy-four (74) patients were studied. A 47,3% had mood disorders, and 43,2% suffered schizophrenia. At discharge from the unit, statistically significant differences were found in all variables analyzed. The improvement in nutritional status, according to the MNA questionnaire, occurred both in Scale A (difference in mean values: -2,23; CI95%: -2,67, -1,79) and in Scale B (difference in mean values: -1,54; CI95%: -2,02, -1,05). CONCLUSIONS: the NIC Nursing Intervention «Management of Nutrition», as the core of the nutritional program, has contributed to improve the nutritional status of hospitalized patients, and this has been reflected in the improvement in nutritional status according to the MNA questionnaire, in the reduction of the level of dependence regarding diet, and in the achievement of associated NOC objectives

Direct detection of protein biomarkers in human fluids using site-specific antibody immobilization strategies.

Design of an optimal surface biofunctionalization still remains an important challenge for the application of biosensors in clinical practice and therapeutic follow-up. Optical biosensors offer real-time monitoring and highly sensitive label-free analysis, along with great potential to be transferred to portable devices. When applied in direct immunoassays, their analytical features depend strongly on the antibody immobilization strategy. A strategy for correct immobilization of antibodies based on the use of ProLinker™ has been evaluated and optimized in terms of sensitivity, selectivity, stability and reproducibility. Special effort has been focused on avoiding antibody manipulation, preventing nonspecific adsorption and obtaining a robust biosurface with regeneration capabilities. ProLinker™-based approach has demonstrated to fulfill those crucial requirements and, in combination with PEG-derivative compounds, has shown encouraging results for direct detection in biological fluids, such as pure urine or diluted serum. Furthermore, we have implemented the ProLinker™ strategy to a novel nanoplasmonic-based biosensor resulting in promising advantages for its application in clinical and biomedical diagnosis.

Trends and challenges of refractometric nanoplasmonic biosensors: a review.

Motivated by potential benefits such as sensor miniaturization, multiplexing opportunities and higher sensitivities, refractometric nanoplasmonic biosensing has profiled itself in a short time span as an interesting alternative to conventional Surface Plasmon Resonance (SPR) biosensors. This latter conventional sensing concept has been subjected during the last decades to strong commercialization, thereby strongly leaning on well-developed thin-film surface chemistry protocols. Not surprisingly, the examples found in literature based on this sensing concept are generally characterized by extensive analytical studies of relevant clinical and diagnostic problems. In contrast, the more novel Localized Surface Plasmon Resonance (LSPR) alternative finds itself in a much earlier, and especially, more fundamental stage of development. Driven by new fabrication methodologies to create nanostructured substrates, published work typically focuses on the novelty of the presented material, its optical properties and its use - generally limited to a proof-of-concept - as a label-free biosensing scheme. Given the different stages of development both SPR and LSPR sensors find themselves in, it becomes apparent that providing a comparative analysis of both concepts is not a trivial task. Nevertheless, in this review we make an effort to provide an overview that illustrates the progress booked in both fields during the last five years. First, we discuss the most relevant advances in SPR biosensing, including interesting analytical applications, together with different strategies that assure improvements in performance, throughput and/or integration. Subsequently, the remaining part of this work focuses on the use of nanoplasmonic sensors for real label-free biosensing applications. First, we discuss the motivation that serves as a driving force behind this research topic, together with a brief summary that comprises the main fabrication methodologies used in this field. Next, the sensing performance of LSPR sensors is examined by analyzing different parameters that can be invoked in order to quantitatively assess their overall sensing performance. Two aspects are highlighted that turn out to be especially important when trying to maximize their sensing performance, being (1) the targeted functionalization of the electromagnetic hotspots of the nanostructures, and (2) overcoming inherent negative influence that stem from the presence of a high refractive index substrate that supports the nanostructures. Next, although few in numbers, an overview is given of the most exhaustive and diagnostically relevant LSPR sensing assays that have been recently reported in literature, followed by examples that exploit inherent LSPR characteristics in order to create highly integrated and high-throughput optical biosensors. Finally, we discuss a series of considerations that, in our opinion, should be addressed in order to bring the realization of a stand-alone LSPR biosensor with competitive levels of sensitivity, robustness and integration (when compared to a conventional SPR sensor) much closer to reality.

Indirect competitive immunoassay for the detection of fungicide Thiabendazole in whole orange samples by Surface Plasmon Resonance.

A highly sensitive and specific SPR-based competitive immunoassay for the detection of Thiabendazole (TBZ) has been developed. An indirect format where a TBZ-protein conjugate is immobilized onto gold surfaces has been selected. Under the optimal conditions, a LOD of 0.67 nM (0.13 µg L(-1)) and an IC(50) of 3.2 nM (0.64 µg L(-1)) have been achieved which are comparable to the values obtained by conventional ELISA. Analysis of real samples has been attempted by first evaluating the influence of complex matrix samples coming from whole oranges and secondly measuring samples containing TBZ previously evaluated by chromatographic methods. A methanolic extraction procedure followed by a simple dilution in assay buffer has proven to be sufficient to measure orange samples using the developed immunoassay with an excellent recovery percentage. The sensitivity and the feasibility of measuring whole orange samples demonstrate the effectiveness and robustness of the SPR biosensor, which can be useful for the determination of TBZ in food at concentrations below the Maximum Residue Levels (MRLs) established by the European legislation.

Guiding light in monolayers of sparse and random plasmonic meta-atoms.

Encouraged by the capacity of surface plasmons to confine and propagate electromagnetic fields, waveguiding concepts have been developed, including combinations of continuous metal films or ordered arrays of metal nanoparticles. So far, waveguiding in the latter systems has been based on near-field or diffractive coupling. Herein, we show that monolayers of sparse and disordered gold nanoparticles support a novel transverse-electric guided mode that, contrary to previous work, relies on the strong enhancement of the polarizability upon excitation of the nanoparticle LSPR, creating an effective refractive index sufficiently high to support light guidance over a large range of frequencies. Excitation of this guided mode offers interesting nanophotonics features and applications such as a tunable total absorption spectral band, attractive for light harvesting applications, or the generation of a large amplification of the sensitivity to changes of refractive index accompanied with striking enhancement of the limit of detection in real biosensing experiments.