A novel biomimetic nanoplasmonic sensor for rapid and accurate evaluation of checkpoint inhibitor immunotherapy.

Immune checkpoint inhibitors (ICIs) emerged as promising immunotherapies for cancer treatment, harnessing the patient's immune system to fight and eliminate tumor cells. However, despite their potential and proven efficacies, checkpoint inhibitors still face important challenges such as the tumor heterogeneity and resistance mechanisms, and the complex in vitro testing, which limits their widespread applicability and implementation to treat cancer. To address these challenges, we propose a novel analytical technique utilizing biomimetic label-free nanoplasmonic biosensors for rapid and reliable screening and evaluation of checkpoint inhibitors. We have designed and fabricated a low-density nanostructured plasmonic sensor based on gold nanodisks that enables the direct formation of a functional supported lipid bilayer, which acts as an artificial cell membrane for tumor ligand immobilization. With this biomimetic scaffold, our biosensing approach provides real-time, highly sensitive analysis of immune checkpoint pathways and direct assessment of the blocking effects of monoclonal antibodies in less than 20 min/test. We demonstrate the accuracy of our biomimetic sensor for the study of the programmed cell death protein 1 (PD1) checkpoint pathway, achieving a limit of detection of 6.7 ng/mL for direct PD1/PD-L1 interaction monitoring. Besides, we have performed dose-response inhibition curves for an anti-PD1 monoclonal antibody, obtaining a half maximal inhibitory concentration (IC50) of 0.43 nM, within the same range than those obtained with conventional techniques. Our biomimetic sensor platform combines the potential of plasmonic technologies for rapid label-free analysis with the reliability of cell-based assay in terms of ligand mobility. The biosensor is integrated in a compact user-friendly device for the straightforward implementation in biomedical and pharmaceutical laboratories.

One-Step and Real-Time Detection of microRNA-21 in Human Samples for Lung Cancer Biosensing Diagnosis.

The rapid diagnosis of cancer, especially in its early stages, is crucial for on-time medical treatment and for increasing the patient survival rate. Lung cancer shows the highest mortality rate and the lowest 5-year survival rate due to the late diagnosis in advanced cancer stages. Providing rapid and reliable diagnostic tools is a top priority to address the problem of a delayed cancer diagnosis. We introduce a nanophotonic biosensor for the direct and real-time detection in human plasma of the microRNA-21-5p biomarker related to lung cancer. The biosensor employs a silicon photonic bimodal interferometric waveguide that provides a highly sensitive detection in a label-free format. We demonstrate a very competitive detectability for direct microRNA-21-5p biomarker assays in human plasma samples (estimated LOD: 25 pM). The diagnostic capability of our biosensor was validated by analyzing 40 clinical samples from healthy individuals and lung cancer patients, previously analyzed by reverse-transcription quantitative polymerase chain reaction (qRT-PCR). We could successfully identify and quantify the levels of microRNA in a one-step assay, without the need for DNA extraction or amplification steps. The study confirmed the significance of implementing this biosensor technique compared to the benchmarking molecular analysis and showed excellent agreement with previous results employing the traditional qRT-PCR. This work opens new possibilities for the true implementation of point-of-care biosensors that enable fast, simple, and efficient early diagnosis of cancer diseases.

Label-Free Plasmonic Biosensor for Rapid, Quantitative, and Highly Sensitive COVID-19 Serology: Implementation and Clinical Validation.

Serological tests are essential for the control and management of COVID-19 pandemic (diagnostics and surveillance, and epidemiological and immunity studies). We introduce a direct serological biosensor assay employing proprietary technology based on plasmonics, which offers rapid (<15 min) identification and quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in clinical samples, without signal amplification. The portable plasmonic device employs a custom-designed multiantigen (RBD peptide and N protein) sensor biochip and reaches detection limits in the low ng mL-1 range employing polyclonal antibodies. It has also been implemented employing the WHO-approved anti-SARS-CoV-2 immunoglobulin standard. A clinical validation with COVID-19 positive and negative samples (n = 120) demonstrates its excellent diagnostic sensitivity (99%) and specificity (100%). This positions our biosensor as an accurate and easy-to-use diagnostics tool for rapid and reliable COVID-19 serology to be employed both at laboratory and decentralized settings for the disease management and for the evaluation of immunological status during vaccination or treatment.

Biochemistry strategies for label-free optical sensor biofunctionalization: advances towards real applicability.

Label-free biosensors, and especially those based on optical transducers like plasmonic or silicon photonic systems, have positioned themselves as potential alternatives for rapid and highly sensitive clinical diagnostics, on-site environmental monitoring, and for quality control in foods or other industrial applications, among others. However, most of the biosensor technology has not yet been transferred and implemented in commercial products. Among the several causes behind that, a major challenge is the lack of standardized protocols for sensor biofunctionalization. In this review, we summarize the most common methodologies for sensor surface chemical modification and bioreceptor immobilization, discussing their advantages and limitations in terms of analytical sensitivity and selectivity, reproducibility, and versatility. Special focus is placed on the suggestions of innovative strategies towards antifouling and biomimetic functional coatings to boost the applicability and reliability of optical biosensors in clinics and biomedicine. Finally, a brief overview of research directions in the area of device integration, automation, and multiplexing will give a glimpse of the future perspectives for label-free optical biosensors.

Design and characterization of high-affinity synthetic peptides as bioreceptors for diagnosis of cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is one of the illnesses caused by Leishmania parasite infection, which can be asymptomatic or severe according to the infecting Leishmania strain. CL is commonly diagnosed by directly detecting the parasites or their DNA in tissue samples. New diagnostic methodologies target specific proteins (biomarkers) secreted by the parasite during the infection process. However, specific bioreceptors for the in vivo or in vitro detection of these novel biomarkers are rather limited in terms of sensitivity and specificity. For this reason, we here introduce three novel peptides as bioreceptors for the highly sensitive and selective identification of acid phosphatase (sAP) and proteophosphoglycan (PPG), which have a crucial role in leishmaniasis infection. These high-affinity peptides have been designed from the conservative domains of the lectin family, holding the ability to interact with the biological target and produce the same effect than the original protein. The synthetic peptides have been characterized and the affinity and kinetic constants for their interaction with the targets (sAP and PPG) have been determined by a surface plasmon resonance biosensor. Values obtained for KD are in the nanomolar range, which is comparable to high-affinity antibodies, with the additional advantage of a high biochemical stability and simpler production. Pep2854 exhibited a high affinity for sAP (KD = 1.48 nM) while Pep2856 had a good affinity for PPG (KD 1.76 nM). This study evidences that these peptidomimetics represent a novel alternative tool to the use of high molecular weight proteins for biorecognition in the diagnostic test and biosensor devices for CL.

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.

Coherent silicon photonic interferometric biosensor with an inexpensive laser source for sensitive label-free immunoassays.

Over the past two decades, integrated photonic sensors have been of major interest to the optical biosensor community due to their capability to detect low concentrations of molecules with label-free operation. Among these, interferometric sensors can be read-out with simple, fixed-wavelength laser sources and offer excellent detection limits but can suffer from sensitivity fading when not tuned to their quadrature point. Recently, coherently detected sensors were demonstrated as an attractive alternative to overcome this limitation. Here we show, for the first time, to the best of our knowledge, that this coherent scheme provides sub-nanogram per milliliter limits of detection in C-reactive protein immunoassays and that quasi-balanced optical arm lengths enable operation with inexpensive Fabry-Perot-type lasers sources at telecom wavelengths.

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.

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.

Site-Specific mRNA Cleavage for Selective and Quantitative Profiling of Alternative Splicing with Label-Free Optical Biosensors.

Alternative splicing of mRNA precursors is a key process in gene regulation, contributing to the diversity of proteomes by the alternative selection of exonic sequences. Alterations in this mechanism are associated with most cancers, enhancing their proliferation and survival, and can be employed as cancer biomarkers. Label-free optical biosensors are ideal tools for the highly sensitive and label-free analysis of nucleic acids. However, their application for alternative splicing analysis has been hampered due to the formation of complex and intricate long-range base-pairing interactions which make the direct detection in mRNA isoforms difficult. To solve this bottleneck, we introduce a methodology for the generation of length-controlled RNA fragments from purified total RNA, which can be easily detected by the biosensor. The methodology seizes RNase H enzyme activity to degrade the upstream and downstream RNA segments flanking the target sequence upon hybridization to specific DNA oligos. It allows the fast and direct monitoring of Fas gene alternative splicing in real time, employing a surface plasmon resonance biosensor. We demonstrate the selective and specific detection of mRNA fragments in the pM-nM concentration range, reducing quantification errors and showing 81% accuracy when compared to RT-qPCR. The site-specific cleavage outperformed random RNA hydrolysis by increasing the detection accuracy by 20%, making this methodology particularly appropriate for label-free quantification of alternative splicing events in complex samples.

Advances in nanoplasmonic biosensors for clinical applications.

Biomarkers are unquestionable biological indicators for diagnosis and therapeutic interventions providing appropriate classification of a wide range of health disorders and risk factors. Nonetheless, the detection and quantification of biomarkers need to be tested with sufficient reliability by robust analytical methods in order to assure clinical performance in health care settings. Since the analytical performance is determined by the sensitivity and specificity of the method employed, techniques have been intensively refined in order to avoid the misinterpretation of results and undesirable bias. Although biomarkers can be detected with the existing analytical techniques, to reproducibly quantify them in decentralized settings or remote locations with the required accuracy is still a challenge. Currently, only a few point-of-care devices for biomarker evaluation are commercially available. Thus, more focused research efforts are needed to overcome these limitations in order to provide universal patient-centered care platforms. To this end, plasmonic biosensors can be conveniently used as portable diagnostic devices for attaining timely and cost-effective clinical outcomes. The development of enhanced performance based on nanoplasmonics technology opens the way for sensor miniaturization, multiplexing and point of care testing. This review covers recent advances and applications of plasmonic and nanoplasmonic biosensors intended for biomarker diagnosis in clinical practice, including cancer, cardiovascular and neurodegenerative diseases. The review specially focuses on: (i) recent progress in plasmonics development including the design of singular nanostructured surfaces, (ii) novel chemical functionalization strategies for the appropriate incorporation of bioreceptors and (iii) plasmonic applications as real operative devices in the clinical field. Future prospects in the use of nanoplasmonic sensor platforms for personalised quantification and management of biomarkers directly in body fluids will also be discussed.

Trimodal Waveguide Demonstration and Its Implementation as a High Order Mode Interferometer for Sensing Application.

This work implements and demonstrates an interferometric transducer based on a trimodal optical waveguide concept. The readout signal is generated from the interference between the fundamental and second-order modes propagating on a straight polymer waveguide. Intuitively, the higher the mode order, the larger the fraction of power (evanescent field) propagating outside the waveguide core, hence the higher the sensitivity that can be achieved when interfering against the strongly confined fundamental mode. The device is fabricated using the polymer SU-8 over a SiO2 substrate and shows a free spectral range of 20.2 nm and signal visibility of 5.7 dB, reaching a sensitivity to temperature variations of 0.0586 dB/ ∘ C. The results indicate that the proposed interferometer is a promising candidate for highly sensitive, compact and low-cost photonic transducer for implementation in different types of sensing applications, among these, point-of-care.

Optimizing the Limit of Detection of Waveguide-Based Interferometric Biosensor Devices.

Waveguide-based photonic sensors provide a unique combination of high sensitivity, compact size and label-free, multiplexed operation. Interferometric configurations furthermore enable a simple, fixed-wavelength read-out making them particularly suitable for low-cost diagnostic and monitoring devices. Their limit of detection, i.e., the lowest analyte concentration that can be reliably observed, mainly depends on the sensors response to small refractive index changes, and the noise in the read-out system. While enhancements in the sensors response have been extensively studied, noise optimization has received much less attention. Here we show that order-of-magnitude enhancements in the limit of detection can be achieved through systematic noise reduction, and demonstrate a limit of detection of ∼ 10 - 8 RIU with a silicon nitride sensor operating at telecom wavelengths.

Sensitive and label-free detection of miRNA-145 by triplex formation.

The development of new strategies for detecting microRNAs (miRNAs) has become a crucial step in the diagnostic field. miRNA profiles depend greatly on the sample and the analytical platform employed, leading sometimes to contradictory results. In this work, we study the use of modified parallel tail-clamps to detect a miRNA sequence involved in tumor suppression by triplex formation. Thermal denaturing curves and circular dichroism (CD) measurements have been performed to confirm that parallel clamps carrying 8-aminoguanine form the most stable triplex structures with their target miRNA. The modified tail-clamps have been tested as bioreceptors in a surface plasmon resonance (SPR) biosensor for the detection of miRNA-145. The detection limit was improved 2.4 times demonstrating that a stable triplex structure is formed between target miRNA and 8-aminoguanine tail-clamp bioreceptor. This new approach is an essential step toward the label-free and reliable detection of miRNA signatures for diagnostic purposes.

Last Advances in Silicon-Based Optical Biosensors.

We review the most important achievements published in the last five years in the field of silicon-based optical biosensors. We focus specially on label-free optical biosensors and their implementation into lab-on-a-chip platforms, with an emphasis on developments demonstrating the capability of the devices for real bioanalytical applications. We report on novel transducers and materials, improvements of existing transducers, new and improved biofunctionalization procedures as well as the prospects for near future commercialization of these technologies.

Study of a low-cost trimodal polymer waveguide for interferometric optical biosensors.

A novel evanescent wave biosensor based on modal interaction between the fundamental mode and the second order mode is proposed and numerically demonstrated. By taking advantage of their symmetries, it is possible to design a device where only the fundamental and the second order modes can propagate, without excitation of the first order mode. With this selection of modes it is possible to achieve a high sensitivity behavior in the biosensor configuration, due to the strong interaction between the evanescent field and the outer surface as compared to previous evanescent wave-based biosensor designs.

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.

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.

Site-directed antibody immobilization using a protein A-gold binding domain fusion protein for enhanced SPR immunosensing.

We have implemented a novel strategy for the oriented immobilization of antibodies onto a gold surface based on the use of a fusion protein, the protein A-gold binding domain (PAG). PAG consists of a gold binding peptide (GBP) coupled to the immunoglobulin-binding domains of staphylococcal protein A. This fusion protein provides an easy and fast oriented immobilization of antibodies preserving its native structure, while leaving the antigen binding sites (Fab) freely exposed. Using this immobilization strategy, we have demonstrated the performance of the immunosensing of the human Growth Hormone by SPR. A limit of detection of 90 ng mL(-1) was obtained with an inter-chip variability lower than 7%. The comparison of this method with other strategies for the direct immobilization of antibodies over gold surfaces has showed the enhanced sensitivity provided by the PAG approach.