A 3D-printed hollow microneedle-based electrochemical sensing device for in situ plant health monitoring.

Plant health monitoring is devised as a new concept to elucidate in situ physiological processes. The need for increased food production to nourish the growing global population is inconsistent with the dramatic impact of climate change, which hinders crop health and exacerbates plant stress. In this context, wearable sensors play a crucial role in assessing plant stress. Herein, we present a low-cost 3D-printed hollow microneedle array (HMA) patch as a sampling device coupled with biosensors based on screen-printing technology, leading to affordable analysis of biomarkers in the plant fluid of a leaf. First, a refinement of the 3D-printing method showed a tip diameter of 25.9 ± 3.7 µm with a side hole diameter on the microneedle of 228.2 ± 18.6 µm using an affordable 3D printer (<500 EUR). Notably, the HMA patch withstanded the forces exerted by thumb pressing (i.e. 20-40 N). Subsequently, the holes of the HMA enabled the fluid extraction tested in vitro and in vivo in plant leaves (i.e. 13.5 ± 1.1 µL). A paper-based sampling strategy adapted to the HMA allowed the collection of plant fluid. Finally, integrating the sampling device onto biosensors facilitated the in situ electrochemical analysis of plant health biomarkers (i.e. H2O2, glucose, and pH) and the electrochemical profiling of plants in five plant species. Overall, this electrochemical platform advances precise and versatile sensors for plant health monitoring. The wearable device can potentially improve precision farming practices, addressing the critical need for sustainable and resilient agriculture in changing environmental conditions.

Water-assisted synthesis of stable and multicolored CsPbX3@SiO2 core-shell nanoparticles as fluorescent probes for biosensing.

Colloidal lead halide perovskite nanocrystals are highly luminescent materials with great promise as fluorescent probes in biosensing as long as their intrinsic instability in aqueous media is effectively addressed. In this study, we successfully prepared stable and multicolored CsPbX3@SiO2 (X = Cl/Br, Br and I) core-shell nanoparticles through a simple method based on the water-induced transformation of Cs4PbX6 into CsPbX3, combined with sol-gel procedures. We observed that the concentration of the Cs4PbX6 precursor plays a crucial role in the formation of isolated nanospheres with uniform silica coating and in controlling the number of core-free particles. Furthermore, our research expands this approach to other halide compositions, resulting in multicolored core-shell nanoparticles with emission wavelengths ranging from 490 to 700 nm, average sizes below 30 nm, and photoluminescence quantum yields close to 60%. Unlike in previous reports, the silica coating boosts the photoluminescence quantum yields compared to uncoated counterparts and provides increased structural stability for more than four days. Moreover, a controlled thermal treatment confers water stability to the as-synthesized nanoparticles. To establish the feasibility of the developed materials as fluorescent probes, we successfully demonstrated their specific recognition of a humanized antibody (omalizumab) used in treating patients with severe allergic asthma. This work paves the way to develop in vitro tests using CsPbX3@SiO2 core-shell nanoparticles as fluorogenic probes.

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.

Exploring the potential of paper-based electrokinetic phenomena in PoC biosensing.

In order to decentralize health care, the development of point-of-care (PoC) assays has gained significant attention in recent decades. The lateral flow immunoassay (LFIA) has emerged as a promising bioanalytical method due to its low cost and single-step detection process. However, its limited sensitivity and inability to detect disease biomarkers at clinically relevant levels have hindered its application for early diagnosis. This review explores the potential of merging different electrokinetic phenomena into paper-based assays to enhance their analytical performance, offering a versatile and affordable approach for PoC testing. The review exposes the challenges faced in integrating electrokinetic phenomena with paper-based biosensing and concludes by discussing the issues that need to be improved to maximize the potential of this technology for early diagnosis.

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.

Simultaneous quantification of six major allergens in commercial foods for children using a multiplex array on a digital versatile disc.

The hypothesis of this study is centered around the logic that an enhanced analysis of potential allergens during the food production can lead to increased accuracy and reliability of food labeling. The development of a cost-effective and straightforward optoelectrical microanalytical system for the simultaneous quantification of the six most common food allergens (peanut, hazelnut, almond, milk, wheat, and soybean) is presented. The system uses a regular versatile disc (DVD) functionalized with highly selective antibodies in a microarray format and a DVD drive as the optical detector. The multiplexed assay reliably (RSD < 20 %) determines the level of the allergenic proteins ranging from 0.1 to 143.4 ng mL-1. The analysis of food consumables (biscuits, seafood substitutes, and probiotic foods) revealed a 100 % accuracy in identifying the allergens ingredients declared on the label. The method offers potential for application as a high throughput biosensing tool for screening multiple allergens in commercial foods.

Lateral Flow Microimmunoassay (LFµIA) for the Reliable Quantification of Allergen Traces in Food Consumables.

Quality assurance and food safety are of great concern within the food industry because of unknown quantities of allergens often present in food. Therefore, there is an ongoing need to develop rapid, sensitive, and easy to use methods that serve as an alternative to mass spectrometry and enzyme-linked immunosorbent assay (ELISA) for monitoring food safety. Lateral flow immunoassay is one of the most used point-of-need devices for clinical, environmental, and food safety applications. Compared to traditional methods, it appears to be a simple and fast alternative for detecting food allergens. However, its reliability is frequently questioned due to the lack of quantitative information. In this study, a lateral flow microimmunoassay (LFµIA) is presented that integrates up to 36 spots in microarray format in a single strip, providing semi-quantitative information about the level of allergens, positive and negative controls, internal calibration, and hook effect. The LFµIA has been evaluated for the on-site simultaneous and reliable quantification of almond and peanut allergens as a proof of concept, demonstrating high sensitivity (185 and 229 µg/kg, respectively), selectivity (77%), and accuracy (RSD 5-25%) when analyzing commercial allergen-suspicious food consumables.

An ultra-sensitive homologous chemiluminescence immunoassay to tackle penicillin allergy.

Penicillin is one of the most widely used antibiotics to treat bacterial infections in clinical practice. The antibiotic undergoes degradation under physiological conditions to produce reactive compounds that in vivo bind self-proteins. These conjugates might elicit an immune response and trigger allergic reactions challenging to diagnose due to the complex immunogenicity. Penicillin allergy delabeling initiatives are now part of antibiotic stewardship programs and include the use of invasive and risky in vivo tests. Instead, the in vitro quantification of specific IgE is highly useful to confirm immediate allergy to penicillins. However, discrepant results associated with the low sensitivity and accuracy of penicillin allergy in vitro tests have limited their routine diagnostic use for delabeling purposes. We aimed to develop a homologous chemiluminescence-based immunochemical method for the reliable determination of specific IgE to penicillin G, using unprecedented synthetic human-like standards. The synthetic standard targets the major antigenic determinant of penicillin G and the paratope of Omalizumab, acting as human-like specific IgE. It is a potent calibrator, highly stable, easy, and inexpensive to produce, overcoming the limitations of the pooled human serum preparations. The developed method achieved a good agreement and strong positive relationship, reaching a detection limit below 0.1 IU mL-1 and excellent reproducibility (RSD <9%). The clinical sensitivity of the assay significantly increased (66%), doubling the accuracy of the reference method with an overall specificity of 100%. The new diagnostic strategy compares favorably with results obtained by the standard procedure, paving the way towards the standardization of penicillin allergy testing, and enhancing the detection sensitivity of specific IgE in serum to tackle reliably ß-lactam allergy delabeling.

An all-in-one point-of-care testing device for multiplexed detection of respiratory infections.

The impact of the COVID-19 pandemic has reinforced the need for rapid, cost-effective, and reliable point-of-care testing (POCT) devices for massive population screening. The co-circulation of SARS-CoV-2 with several seasonal respiratory viruses highlights the need for multiplexed biosensing approaches. Herein, we present a fast and robust all-in-one POCT device for parallel viral antigen and serological analysis. The biosensing approach consists of a functionalized polycarbonate disc-shaped surface with microfluidic structures, where specific bioreagents are immobilized in microarray format, and a portable optoelectronic analyzer. The biosensor quantifies the concentration of viral antigens and specific immunoglobulins G and M for SARS-CoV-2, influenza A/B, adenovirus, and respiratory syncytial virus, using 30 µL of a sample. The semi-automated analysis of 6 samples is performed in 30 min. Validation studies performed with 135 serum samples and 147 nasopharyngeal specimens reveal high diagnostic sensitivity (98-100%) and specificity (84-98%), achieving an excellent agreement (κ = 0.937) with commercial immunoassays, which complies with the World Health Organization criteria for POC COVID-19 diagnostic tests. The versatility of the POCT device paves the way for the detection of other pathogens and analytes in the incoming post-pandemic world, integrating specific bioreagents against different variants of concerns and interests.

Synthesis of skeletally diverse ß-lactam haptens for the in vitro diagnosis of IgE-mediated drug allergy.

We present the first synthesis of ß-lactam-derived haptens, leveraging the principles of diversity-oriented synthesis to discover compounds for drug allergy in vitro testing. We designed, synthesised, and performed in vitro immunological evaluation on 18 structurally diverse haptens derived from ß-lactam antibiotics. The antigens obtained with the synthesised haptens allow for the detection of specific anti-ß-lactam immunoglobulins G and E. Excellent diagnostic sensitivity (83%) and specificity (100%) were achieved when the panel of antigens was tested against a cohort of 31 human serum samples using a multiplexed compact disc-based in vitro testing tool. We posit that adopting this strategy could aid ß-lactam delabeling initiatives.

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.

Multiplexed analytical approaches to beta-lactam allergy in vitro testing standardization.

The suspicion of beta-lactam allergy directly contributes to the prescription of antibiotics that diverge from the guidelines, increasing antimicrobial resistance, one of the biggest threats to global health. In vitro quantification of specific IgE is very useful for monitoring allergy, as it confirms or rules out immediate beta-lactam drug allergy and helps find safe alternative antibiotic stewardship. However, reliable in vitro quantification of specific IgE to beta-lactam antibiotics by immunoassay is challenging because of the difficulty of having selective immunoreagents, mainly beta-lactam antigens, and its low concentration levels in serum. Thus, reliable and sensitive in vitro tests for multiplex detection of allergy to different beta-lactam antibiotics is currently essential for clinical diagnosis. Nevertheless, the lack of standardization of quantitative in vitro methods makes the comparison and interpretation of the results difficult. Here, as proof of concept, we report an improved multiplex microimmunoassay for beta-lactam allergy in vitro testing standardization. The results revealed that homologous calibration allows reliable quantification of specific IgE in human serum at very low concentrations (144 ng L-1). Moreover, the reproducibility of the results increases 2-fold using an internal standard, achieving accurate quantitative information: 93% and 106% recovery for penicillin and amoxicillin, respectively. We simultaneously evaluated the reliability of the improved multiplexed in vitro method in a cohort of 40 human serum samples and achieved excellent agreement (0.99) with a currently used in vitro test.

Boosting the sensitivity of in vitroß-lactam allergy diagnostic tests.

The synthesis of structurally new haptens and the development of suitable antigens are essential for boosting the sensitivity of drug allergy diagnostic testing. Unprecedented structural antigens for benzylpenicillin and amoxicillin are characterised and evaluated in a cohort of 70 subjects with a turnkey solution based on consumer electronics.

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.

Neo-antigens for the serological diagnosis of IgE-mediated drug allergic reactions to antibiotics cephalosporin, carbapenem and monobactam.

New antigens deriving from -lloyl and -llanyl, major and minor determinants, respectively, were produced for ß-lactam antibiotics cefuroxime, cefotaxime, ceftriaxone, meropenem and aztreonam. Twenty ß-lactam antigens were produced using human serum albumin and histone H1 as carrier proteins. Antigens were tested by multiplex in vitro immunoassays and evaluated based on the detection of specific IgG and IgE in the serum samples. Both major and minor determinants were appropriate antigens for detecting specific anti-ß-lactam IgG in immunised rabbit sera. In a cohort of 37 allergic patients, we observed that only the minor determinants (-llanyl antigens) were suitable for determining specific anti-ß-lactam IgE antibodies with high sensitivity (< 0.01 IU/mL; 24 ng/L) and specificity (100%). These findings reveal that not only the haptenisation of ß-lactam antibiotics renders improved molecular recognition events when the 4-member ß-lactam ring remains unmodified, but also may contribute to develop promising minor antigens suitable for detecting specific IgE-mediated allergic reactions. This will facilitate the development of sensitive and selective multiplexed in vitro tests for drug-allergy diagnoses to antibiotics cephalosporin, carbapenem and monobactam.

Highly sensitive optoelectrical biosensor for multiplex allergy diagnosis.

Compact multiplexed biosensors systems hold great potential for diagnosis of diseases where the detection of multiple biomarkers is required. Hypersensitivity Immunoglobulin E mediated syndromes are primary immunodeficiency disorders associated with sensitization to allergens. Assessing immunoglobulin E (IgE) sensitization to allergens is an important strategy for allergy diagnosis. Here, we report for the first time a reliable, flexible and cost-effective optoelectrical biosensor system for the simultaneous determination of total and allergen-specific IgE and IgG, antibodies using an immunogold-silver signal amplification method. The biosensor was constructed on a regular digital versatile disc (DVD) to immobilize a panel of 12 allergen extracts or pure proteins in microarray format, as a proof of concept. The multiplexed biosensor showed a limit of detection of 0.26 IU/mL (624 pg/mL) and 14 ng/mL for IgE and IgG antibodies, respectively. The system was successfully applied in a cohort of 127 human serum samples, showing good sensitivity (97.6%) as well as specificity (85.7%), and an excellent area under the curve (AUC) value was found at 0.977 (confidence interval, CI 0.957 to 0.990) as compared and validated with a reference clinical immunofluorescence assay, confirming an excellent correlation between both techniques. The multiplex biosensor system with on-demand panel composition can be used fully autonomously in clinical or mobile laboratory settings without the need for any additional medical equipment, with which could make it suitable for massive allergy screening campaigns to better define sensitization profiles.

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.

Detecting Nonvolatile Life- and Nonlife-Derived Organics in a Carbonaceous Chondrite Analogue with a New Multiplex Immunoassay and Its Relevance for Planetary Exploration.

Potential martian molecular targets include those supplied by meteoritic carbonaceous chondrites such as amino acids and polycyclic aromatic hydrocarbons and true biomarkers stemming from any hypothetical martian biota (organic architectures that can be directly related to once living organisms). Heat extraction and pyrolysis-based methods currently used in planetary exploration are highly aggressive and very often modify the target molecules making their identification a cumbersome task. We have developed and validated a mild, nondestructive, multiplex inhibitory microarray immunoassay and demonstrated its implementation in the SOLID (Signs of Life Detector) instrument for simultaneous detection of several nonvolatile life- and nonlife-derived organic molecules relevant in planetary exploration and environmental monitoring. By utilizing a set of highly specific antibodies that recognize D- or L- aromatic amino acids (Phe, Tyr, Trp), benzo[a]pyrene (B[a]P), pentachlorophenol, and sulfone-containing aromatic compounds, respectively, the assay was validated in the SOLID instrument for the analysis of carbon-rich samples used as analogues of the organic material in carbonaceous chondrites or even Mars samples. Most of the antibodies enabled sensitivities at the 1-10 ppb level and some even at the ppt level. The multiplex immunoassay allowed the detection of B[a]P as well as aromatic sulfones in a water/methanol extract of an Early Cretaceous lignite sample (c.a., 140 Ma) representing type IV kerogen. No L- or D-aromatic amino acids were detected, reflecting the advanced diagenetic stage and the fossil nature of the sample. The results demonstrate the ability of the liquid extraction by ultrasonication and the versatility of the multiplex inhibitory immunoassays in the SOLID instrument to discriminate between organic matter derived from life and nonlife processes, an essential step toward life detection outside Earth.

Mapping molecular binding by means of conformational dynamics measurements.

Protein-protein interactions are key in virtually all biological processes. The study of these interactions and the interfaces that mediate them play a key role in the understanding of biological function. In particular, the observation of protein-protein interactions in their dynamic environment is technically difficult. Here two surface analysis techniques, dual polarization interferometry and quartz crystal microbalance with dissipation monitoring, were paired for real-time mapping of the conformational dynamics of protein-protein interactions. Our approach monitors this dynamics in real time and in situ, which is a great advancement within technological platforms for drug discovery. Results agree with the experimental observations of the interaction between the TRIM21α protein and circulating autoantibodies via a bridging bipolar mechanism. This work provides a new chip-based method to monitor conformational dynamics of protein-protein interactions, which is amenable to miniaturized high-throughput determination.

Simultaneous determination of four food allergens using compact disc immunoassaying technology.

A multiplex competitive microimmunoassay for the simultaneous determination of gliadin, casein, ß-lactoglobulin, and ovalbumin is presented. The assay in microarray format is performed on a DVD where the allergens are physisorbed on the polycarbonate surface of the disc. The immunointeraction is detected using a mixture of specific gold-labeled antibodies and the signal amplified with the silver enhancement method. The optical density of the precipitate, read by a DVD drive, is related to the concentration of the four allergens in sample. An optimized protocol for the simultaneous extraction of the allergen proteins from food samples is also addressed. The suitability of the method is demonstrated for the simultaneous quantitative extraction and determination of the targeted allergens in spiked baby foods, juices, and beers. The sensitivity (EC50) of the multiplexed assay was 0.04, 0.40, 0.08, and 0.16 mg L-1 for gliadin, casein, ß-lactoglobulin, and ovalbumin, respectively, and the recovery results from the analysis of food samples ranged from 72 to 117%. A portable, easy-to-use, array-based bioanalytical method is developed for quantification of food allergens with a limit of detection below the accepted levels of the international legislations, which allows promotion of food safety and quality. Graphical abstract GLI Gliadin, CAS Casein, ß-LAC ß-lactoglobulin, OVA Ovalbumin.