Real-world evaluation of a QCM-based biosensor for exhaled air.

The biosensor, named "virusmeter" in this study, integrates quartz crystal microbalance technology with an immune-functionalized chip to distinguish between symptomatic patients with respiratory diseases and healthy individuals by analyzing exhaled air samples. Renowned for its compact design, rapidity, and noninvasive nature, this device yields results within a 5-min timeframe. Evaluated under controlled conditions with 54 hospitalized symptomatic COVID-19 patients and 128 control subjects, the biosensor demonstrated good overall sensitivity (98.15%, 95% CI 90.1-100.0) and specificity (96.87%, 95% CI 92.2-99.1). This proof-of-concept presents an innovative approach with significant potential for leveraging piezoelectric sensors to diagnose respiratory diseases.

Standardizing In Vitro ß-Lactam Antibiotic Allergy Testing with Synthetic IgE.

The global prevalence of ß-lactam allergy poses a major challenge in administering first-line antibiotics, such as penicillins, to a significant portion of the population. The lack of ß-lactam IgE antibody pools with defined selectivity hampers the standardization and validation of in vitro assays for ß-lactam allergy testing. To address this limitation, this study introduces a synthetic IgE specific to ß-lactam antibiotics as a valuable tool for drug allergy research and diagnostic tests. Using phage display technology, we constructed a library of human single-chain antibody fragments (scFv) to target the primary determinant of amoxicillin, a widely used ß-lactam antibiotic. Subsequently, we produced a complete human synthetic IgE molecule using the highly efficient baculovirus expression vector system. This synthetic IgE molecule served as a standard in an in vitro chemiluminescence immunoassay for ß-lactam antibiotic allergy testing. Our results demonstrated a detection limit of 0.05 IU/mL (0.63 pM), excellent specificity (100%), and a four-fold higher clinical sensitivity (73%) compared to the in vitro reference assay when testing a cohort of 150 serum samples. These findings have significant implications for reliable interlaboratory comparison studies, accurate labeling of allergic patients, and combating the global public health threat of antimicrobial resistance. Furthermore, by serving as a valuable trueness control material, the synthetic IgE facilitates the standardization of diagnostic tests for ß-lactam allergy and demonstrates the potential of utilizing this synthetic strategy as a promising approach for generating reference materials in drug allergy research and diagnostics.

Reversed-phase allergen microarrays on optical discs for multiplexed diagnostics of food allergies.

A high percentage of the population suffers from multiple food allergies justifying  the importance of reliable diagnostic methods. Single-analyte solutions based on the determination of specific immunoglobulins E (sIgE) are safe and fast but are generally time-consuming and expensive. Thus sustainable microanalytical methods that provide multianalyte profiling information are highly demanded. This work presents the in vitro biosensing of specific IgE levels based on a reversed-phase allergen array. The approach consists of optical biosensing supported by direct multiplex immunoassays and on-disc technology. It identifies 12 sIgE associated with food allergies in a single analysis with a low serum sample volume (25 µL). After processing captured images, specific signals for each target biomarker correlate to their concentration. The assay analytically performs well with 0.3 IU/mL and 0.41 IU/mL as the detection and quantification limits in serum, respectively. This novel method achieves excellent clinical specificity (100%) and high sensitivity (91.1%), considering the diagnosis obtained by clinical history and ImmunoCAP analysis. The results demonstrate that microanalytical systems based on allergen arrays can potentially diagnose multiple food allergies and are easily implemented in primary care laboratory settings.

Surface Bragg gratings of proteins patterned on integrated waveguides for (bio)chemical analysis.

The incorporation of biomacromolecules onto silicon waveguiding microstructures constitutes a growing trend that pushes towards compact and miniaturized biosensing systems. This paper presents the integration of one-dimensional periodic nanostructures of proteins on the surface of micrometric silicon waveguides for transducing binding events between biomacromolecules. The study demonstrates this new bioanalytical principle by experimental results and theoretical calculations, and proves that rib waveguides (1--1.6-µm width) together with protein gratings (495--515-nm period) display suitable spectral responses for this optical biosensing system. Protein assemblies of bovine serum albumin are fabricated on the surface of silicon nitride waveguides, characterized by electron microscopy, and their response is measured by optical frequency domain reflectometry along the fabrication process and the subsequent stages of the biorecognition assays. Detection and quantification limits of 0.3 and 3.7 µg·mL-1, respectively, of specific antibodies are inferred from experimental dose-response curves. Among other interesting features, the results of this study point towards new miniaturized and integrated sensors for label-free bioanalysis.

Bispecific Single-Domain Antibodies as Highly Standardized Synthetic Calibrators for Immunodiagnosis.

Commonly, serological immunoassays and diagnostic kits include reference standard reagents (calibrators) that contain specific antibodies to be measured, which are used for the quantification of unknown antibodies present in the sample. However, in some cases, such as the diagnosis of allergies or autoimmune diseases, it is often difficult to have sufficient quantities of these reference standards, and there are limitations to their lot-to-lot reproducibility and standardization over time. To overcome this difficulty, this study introduces the use of surrogate recombinant calibrators formulated on the basis of two single-domain antibodies (nanobodies) combined through a short peptide linker to produce a recombinant bispecific construct. One of the nanobodies binds to the cognate analyte of the target antibody and the second is specific for the paratope of the secondary detecting antibody. The bispecific nanobody inherits the outstanding properties of stability and low-cost production by bacterial fermentation of the parent nanobodies, and once calibrated against the biological reference standard, it can be reproduced indefinitely from its sequence in a highly standardized manner. As a proof of concept, we present the generation and characterization of two bispecific calibrators with potential application for the diagnosis of allergy against the antibiotics aztreonam and amoxicillin in humans.

A genosensor for detecting single-point mutations in dendron chips after blocked recombinase polymerase amplification.

A biosensing system was developed to accurately detect a single nucleotide change in the target organism genome, integrating a selective isothermal amplification and a sensitive dendron-mediated DNA hybridization assay in the array format. The novelty arises from the coupling reactions of the dendron and its use as a crosslinker. The allele-specific probes were oligonucleotide-dendron conjugates prepared by fast and clean click-chemistry (thiol-yne reaction) and coupled onto the photo-activated surface of polycarbonate substrates (carbodiimide reaction). The output was forest-array chips with multipoint-site crosslinkers and compatible with current microarray fabrication technologies. The products of blocked recombinase polymerase amplification (blocked RPA), formed at 37 °C, were hybridized with attached probes for specific nucleotide genotyping. The developed approach exhibited sensitive recognition of DNA variants compared to chips based on linear crosslinkers (10-100 fold), showing excellent analytical performances for planar chip and fluidic formats. The methodology was successfully applied to detect the H1047R mutation in the PIK3CA gene (c.3140A > G) from clinical samples of human cancer tissues, the results being consistent with sequencing techniques. The colorimetric biosensing method was reliable, versatile, low cost, sensitive (detection limit genomic DNA: 0.02 ng µL-1), and specific (accuracy >95%).

Hydrogel-based holographic sensors and biosensors: past, present, and future.

Hydrogel-based holographic sensors consist of a holographic pattern in a responsive hydrogel that diffracts light at different wavelengths depending on the dimensions and refractive index changes in the material. The material composition of hydrogels can be designed to be specifically responsive to different stimuli, and thus the diffraction pattern can correlate with the amount of analyte. According to this general principle, different approaches have been implemented to achieve label-free optical sensors and biosensors, with advantages such as easy fabrication or naked-eye detection. A review on the different approaches, sensing materials, measurement principles, and detection setups, and future perspectives is offered.

Enhanced asymmetric blocked qPCR method for affordable detection of point mutations in KRAS oncogene.

An accurate genetic diagnostic is key for adequate patient management and the suitability of healthcare systems. The scientific challenge lies in developing methods to discriminate those patients with certain genetic variations present in tumor cells at low concentrations. We report a method called enhanced asymmetric blocked qPCR (EAB-qPCR) that promotes the blocker annealing against the primer-template hybrid controlling thermal cycling and reaction conditions with nonmodified oligonucleotides. Real-time fluorescent amplification curves of wild-type alleles were delayed by about eight cycles for EAB-qPCR, compared to conventional blocked qPCR approaches. This method reduced the amplification of native DNA variants (blocking percentage 99.7%) and enabled the effective enrichment of low-level DNA mutations. Excellent performance was estimated for the detection of mutated alleles in sensitivity (up to 0.5% mutant/total DNA) and reproducibility terms, with a relative standard deviation below 2.8%. The method was successfully applied to the mutational analysis of metastatic colorectal carcinoma from biopsied tissues. The determined single-nucleotide mutations in the KRAS oncogene (codon 12-13) totally agreed with those obtained from next-generation sequencing. EAB-qPCR is an accurate cheap method and can be easily incorporated into daily routine to detect mutant alleles. Hence, these features are especially interesting to facilitate the diagnosis and prognosis of several clinical diseases.

Multiparametric Highly Sensitive Chemiluminescence Immunoassay for Quantification of ß-Lactam-Specific Immunoglobulin E.

ß-lactams (BLCs) are the most widely used antibiotics and consequently the most common cause of drug allergy in the world. The diagnosis of drug allergy is complex and represents a serious challenge that includes a wide variety of methods. In vitro tests are based on the immunological determination of allergen-specific IgE, but the tests in the market lack the required sensitivity and specificity. In addition, the large sample volume, long incubation times, and single-plex configuration have brought their use into question to complement the clinical information. Here, we report a chemiluminescence immunoassay (CLIA) for multiparametric quantification of specific IgE to penicillin G, penicillin V, amoxicillin, and piperacillin, using histone H1 as a carrier. The developed CLIA allowed the determination of BLC-specific IgE below 0.1 IU/mL, thus allowing identification of allergic patients with better sensitivity, using only 25 µL of a sample (serum). The immunoassay was successfully applied in a cohort of 140 human serum samples, showing good sensitivity (64.6%) as well as specificity (100%), which significantly improve the predictive character of existing BLC-allergy in vitro tests.

Digital versatile discs as platforms for multiplexed genotyping based on selective ligation and universal microarray detection.

The development of a high-performance assay readout using integrated detectors is a current challenge in the implementation of DNA tests in diagnostic laboratories, particularly for supporting pharmacogenetic tests. A method for allelic discrimination, associated with single nucleotide polymorphisms (SNPs), is presented. Genomic DNA is extracted from blood and buccal swab samples. The procedure comprises fast multiplex ligation-dependent probe amplification, PCR amplification using universal primers and subsequent barcode hybridization. In this last step, each product is recognized by the specific probes immobilized on the surface of an optical disc. Assay results can be obtained with a disc reader. The optical sensing method in a DNA microarray format was optimized and evaluated for the simultaneous identification of 28 polymorphisms associated with psychiatric pharmacogenomics. The target biomarkers were located in the genes related to drug-metabolizing enzymes and drug transporters. The multiplexing capability and assay selectivity strongly depended on correct design (ligation probes, tails and barcodes). The discriminant analysis of reader outputs (spot intensities) led to patients being classified into different allelic populations. The obtained assignations correlated properly with the results provided by the reference technique (bead arrays), and the assay ended in an 8-fold shorter time using affordable equipment. The combination of a highly selective genotyping reaction as array-MLPA and the compact disc technology provides a reliable point-of-care approach. This genotyping tool is useful for the selection of personalized drug therapies in decentralized clinical laboratories.

Photoclick chemistry to create dextran-based nucleic acid microarrays.

In the literature, there are reports of the utilization of various hydrogels to create generic platforms for protein microarray applications. Here, a novel strategy was developed to obtain high-performance microarrays. In it, a dextran hydrogel is used to covalently immobilize oligonucleotides and proteins. This method employs aqueous solutions of dextran methacrylate (Dx-MA), which is a biocompatible photopolymerizable monomer. Capture probes are immobilized inside the hydrogel via a light-induced thiol-acrylate coupling reaction at the same time as the dextran polymer is formed. Hydrogel microarrays based on this technique were prepared on different surfaces, such as a Blu-ray Disk and polycarbonate or alkene-functionalized glass slides, and these systems showed high probe-loading capabilities and good biorecognition yields. This methodology presents advantages such as a low cost, a short analysis time, a low limit of detection, and multiplexing capabilities, among others. Confocal fluorescence microscopy analysis demonstrated that in these hydrogel-based microarrays, receptor immobilization and the biorecognition event occurred within the hydrogel and not merely on the surface.

New structural insights into the role of TROVE2 complexes in the on-set and pathogenesis of systemic lupus erythematosus determined by a combination of QCM-D and DPI.

The mechanism of self-recognition of the autoantigen TROVE2, a common biomarker in autoimmune diseases, has been studied with a quartz crystal microbalance with dissipation monitoring (QCM-D) and dual polarization interferometry (DPI). The complementarity and remarkable analytical features of both techniques has allowed new insights into the onset of systemic lupus erythematosus (SLE) to be achieved at the molecular level. The in vitro study for SLE patients and healthy subjects suggests that anti-TROVE2 autoantibodies may undergo an antibody bipolar bridging. An epitope-paratope-specific binding initially occurs to activate a hidden Fc receptor in the TROVE2 tertiary structure. This bipolar mechanism may contribute to the pathogenic accumulation of anti-TROVE2 autoantibody immune complex in autoimmune disease. Furthermore, the specific calcium-dependent protein-protein bridges point out at how the TRIM21/TROVE2 association might occur, suggesting that the TROVE2 protein could stimulate the intracellular immune signaling via the TRIM21 PRY-SPRY domain. These findings may help to better understand the origins of the specificity and affinity of TROVE2 interactions, which might play a key role in the SLE pathogenesis. This manuscript gives one of the first practical applications of two novel functions (-df/dD and Δh/molec) for the analysis of the data provided by QCM-D and DPI. In addition, it is the first time that QCM-D has been used for mapping hidden Fc receptors as well as linear epitopes in a protein tertiary structure. Graphical abstract ᅟ.

Phosphorylcholine-based hydrogel for immobilization of biomolecules. Application to fluorometric microarrays for use in hybridization assays and immunoassays, and nanophotonic biosensing.

An approach is presented for covalent immobilization of biomolecules on an acrylate phosphorylcholine hydrogel. The immobilization and the hydrogel formation take place simultaneously by a thiol-acrylate coupling reaction, induced by UV-light (254 nm). The hydrogel is prepared on two polymeric surfaces (the HardCoat protective layer of Blu-Ray discs, and SU-8) and applied to fluorescence microarray and label-free interferometric detection. For the first, Cy5 labeled analytes are used (λem 635 nm) and, for the second, a periodic array of high-aspect ratio nanopillars detects unlabeled analytes by interferometry. Bioavailability of the immobilized probes is demonstrated in labeled assays; for the case of oligonucleotides by discriminating single nucleotide polymorphisms, and, for the case of antibodies, by BSA immunorecognition. The raw hydrogel is employed to detect human C-reactive protein, in both labeled and non-labeled assay formats, with sensitivities of 30 ng·mL-1 and 2 pg·mL-1, respectively. Graphical abstract Schematic presentation of the phosphorylcholine (MPC) hydrogel preparation onto BluRay disc and SU-8 nanopillars to perform fluorescence and label-free interferometric detection, respectively. It selectively detects C-reactive protein (CRP), but it can covalently immobilize antibodies or nucleid acid probes to detect other analytes.

Fluor-thiol Photocoupling Reaction for Developing High Performance Nucleic Acid (NA) Microarrays.

Spatially controlled anchoring of NA probes onto microscope glass slides by a novel fluor-thiol coupling reaction is performed. By this UV-initiated reaction, covalent immobilization in very short times (30 s at 254 nm) is achieved with probe densities of up to 39.6 pmol/cm2. Modulating the surface hydrophobicity by combining a hydrophobic silane and a hydrophilic silane allows the fabrication of tuned surfaces where the analyte approaches only the anchored probe, which notably reduces nonspecific adsorption and the background. The generated substrates have proven clear advantages for discriminating single-base-pair mismatches, and for detecting bacterial PCR products. The hybridization sensitivity achieved by these high-performance surfaces is about 1.7 pM. Finally, this anchoring reaction is demonstrated using two additional surfaces: polytetrafluoroethylene (PTFE) and polyvinylidene fluoride (PVDF) membranes. This provides a very interesting pathway for anchoring thiolated biomolecules onto surfaces with C-F motifs via a quick clean UV reaction.

Blocked recombinase polymerase amplification for mutation analysis of PIK3CA gene.

A blocked recombinase polymerase amplification (blocked-RPA) approach has been developed for the enrichment of mutated templates in heterogeneous specimens as tumor tissues. This isothermal amplification technique opens alternative solutions for meeting the technological demand of physician office laboratories. Herein, the detection of mutations in PIK3CA gene, such as p.E545K, and p.H1047L, is presented. The main element was an oligonucleotide (dideoxycytidine functionalized at 3'-end) which matched with wild-type sequence in the target locus. The amplification was performed operating at 37 °C during 40 min. The results demonstrated that the competition between the upstream primer and the blocker reduced the percentage of amplified wild-type allele, making the detection of the present mutation easier. For mutation discrimination, a fast hybridization assay was performed in microarray format on plastic chip and colorimetric detection. This approach enabled the reliable discrimination of specific mutations against a background of up to 95% wild-type DNA. The applicability of the method, based on the combination of blocked-RPA and low-cost chip hybridization, was successfully proven for the genotyping of various cancer cell lines as well as tumor tissues. The assignations agreed with those provided by next-generation sequencing. Therefore, these investigations would support a personalized approach to patient care based on the molecular signature of human cancers.

Modulating receptor-ligand binding in biorecognition by setting surface wettability.

Modulation of support wettability used for microarray format biosensing has led to an improvement of results. Hydrophobicity of glass chips was set by derivatizing with single vinyl organosilanes of different chain length and silane mixtures. Thiol-ene photochemical linking has been used as effective chemistry for covalent anchoring of thiolated probes. Lowest unspecific binding and highest signal intensity and SNR were obtained with large hydrocarbon chain (C22) silanes or a shorter one (C10) containing fluorine atoms. SNR resulting values are improved, reaching levels higher than 1500 in some cases, when using vinyl silanes modified with 1% C10 alkyl fluorinated one, because mild hydrophobicity was achieved (water contact angle ca. 110°) for all silanes, including the short C2 and C3, thus giving rise to smaller and better defined array spots. In addition, unspecific binding of reagents and targets was totally withdrawn. Hence, good-performing surfaces for biosensing applications can be built using appropriate organosilane reagent selection, including fluorinated ones. Graphical abstract ᅟ.

Indirect Microcontact Printing to Create Functional Patterns of Physisorbed Antibodies.

Microcontact printing (µCP) is a practical and versatile approach to create nanostructured patterns of biomolecular probes, but it involves conformational changes on the patterned bioreceptors that often lead to a loss on the biological activity of the resulting structures. Herein we introduce indirect µCP to create functional patterns of bioreceptors on solid substrates. This is a simple strategy that relies on physisorbing biomolecular probes of interest in the nanostructured gaps that result after patterning backfilling agents by standard µCP. This study presents the approach, assesses bovine serum albumin as backfilling agent for indirect µCP on different materials, reports the limitations of standard µCP on the functionality of patterned antibodies, and demonstrates the capabilities of indirect µCP to solve this issue. Bioreceptors were herein structured as diffractive gratings and used to measure biorecognition events in label-free conditions. Besides, as a preliminary approach towards sensing biomarkers, this work also reports the implementation of indirect µCP in an immunoassay to detect human immunoglobulin E.

Experimental Study of the Oriented Immobilization of Antibodies on Photonic Sensing Structures by Using Protein A as an Intermediate Layer.

A proper antibody immobilization on a biosensor is a crucial step in order to obtain a high sensitivity to be able to detect low target analyte concentrations. In this paper, we present an experimental study of the immobilization process of antibodies as bioreceptors on a photonic ring resonator sensor. A protein A intermediate layer was created on the sensor surface in order to obtain an oriented immobilization of the antibodies, which enhances the interaction with the target antigens to be detected. The anti-bovine serum albumin (antiBSA)-bovine serum albumin (BSA) pair was used as a model for our study. An opto-fluidic setup was developed in order to flow the different reagents and, simultaneously, to monitor in real-time the spectral response of the photonic sensing structure. The antiBSA immobilization and the BSA detection, their repeatability, and specificity were studied in different conditions of the sensor surface. Finally, an experimental limit of detection for BSA recognition of only 1 ng/mL was obtained.

Diffractive Protein Gratings as Optically Active Transducers for High-Throughput Label-free Immunosensing.

A novel label-free biosensing approach based on bioreceptor networks patterned as diffractive gratings (biogratings) has been developed. Nanogrooved structures were used as optically active scaffolds for producing arrays of functional BSA biogratings on low energy surfaces by a water-assisted variant of microcontact printing. An analytical scanner, comprising a LightScribe compact disk drive, was developed to measure the diffraction patterns of these biogratings, thus allowing biointeractions to be quantitatively sensed in a multiplex and label-free fashion by means of diffraction efficiency changes. The approach was demonstrated by immunoassaying IgGs, reaching well-correlated responses with quantification and detection limits of 1.3 and 5.2 nM, respectively. These results provide appealing insights into cost-effective, portable, and scalable alternatives for designing new analytical technologies based on diffractive gratings of bioreceptors.

Improved Performance of DNA Microarray Multiplex Hybridization Using Probes Anchored at Several Points by Thiol-Ene or Thiol-Yne Coupling Chemistry.

Nucleic acid microarray-based assay technology has shown lacks in reproducibility, reliability, and analytical sensitivity. Here, a new strategy of probe attachment modes for silicon-based materials is built up. Thus, hybridization ability is enhanced by combining thiol-ene or thiol-yne click chemistry reactions with a multipoint attachment of polythiolated probes. The viability and performance of this approach was demonstrated by specifically determining Salmonella PCR products up to a 20 pM sensitivity level.