Immunoglobulins in COVID-19 pneumonia: from the acute phase to the recovery phase.

BACKGROUND: COVID-19 pneumonia causes hyperinflammatory response that culminates in acute respiratory syndrome (ARDS) related to increased multiorgan dysfunction and mortality risk. Antiviral-neutralizing immunoglobulins production reflect the host humoral status and illness severity, and thus, immunoglobulin (Ig) circulating levels could be evidence of COVID-19 prognosis. METHODS: The relationship among circulating immunoglobulins (IgA, IgG, IgM) and COVID-19 pneumonia was evaluated using clinical information and blood samples in a COVID-19 cohort composed by 320 individuals recruited during the acute phase and followed up to 4 to 8 weeks (n = 252) from the Spanish first to fourth waves. RESULTS: COVID-19 pneumonia development depended on baseline Ig concentrations. Circulating IgA levels together with clinical features at acute phase was highly associated with COVID-19 pneumonia development. IgM was positively correlated with obesity (ρb = 0.156, P = 0.020), dyslipemia (ρb = 0.140, P = 0.029), COPD (ρb = 0.133, P = 0.037), cancer (ρb = 0.173, P = 0.007) and hypertension (ρb = 0.148, P = 0.020). Ig concentrations at recovery phase were related to COVID-19 treatments. CONCLUSIONS: Our results provide valuable information on the dynamics of immunoglobulins upon SARS-CoV-2 infection or other similar viruses.

DNA biosensors based on gold nanoparticles-modified graphene oxide for the detection of breast cancer biomarkers for early diagnosis.

Two different DNA (ERBB2c and CD24c) modified gold nanoparticles and graphene oxide loaded on glassy carbon electrodes were prepared for early detection of breast cancer markers by electrochemical detection of HER2. Comparative study of ERBB2c and CD24c for the detection was carried out. A "sandwich-type" detection strategy was employed in this electrochemical DNA biosensor and its response was measured by amperometric detection. The electrochemical signal enhancement achieved via gold nanoparticles and grapheme oxide system allowed for sensitive detection of the breast cancer biomarker ERBB2 and the control marker CD24. The modified graphene oxide was characterised using Raman spectroscopy, UV-visible spectroscopy, Fourier transform infrared spectroscopy transmission electron microscopy, scanning electron microscopy and energy-dispersive X-ray spectroscopy. The various steps involved in the modification of a glassy carbon electrode with graphene oxide, gold nanoparticles and DNA probes, target and reporter probe were electrochemically characterised using cyclic voltammetry and electrochemical impedance spectroscopy. Using amperometric detection of a horse radish peroxidase label, detection limits of 0.16nM and 0.23nM were obtained with sensitivity 378nA/nM and 219nA/nM for ERBB2 andCD24 respectively.

Revisiting the use of sPLA2-sensitive liposomes in cancer therapy.

The first developed secretory phospholipase A2 (sPLA2) sensitive liposomal cisplatin formulation (LiPlaCis®) is currently undergoing clinical evaluation. In the present study we revisit and evaluate critical preclinical parameters important for the therapeutic potential and safety of platinum drugs, here oxaliplatin (L-OHP), formulated in sPLA2 sensitive liposomes. We show the mole percentage of negatively charged phospholipid needed to obtain enzyme-sensitivity for saturated systems is ≥25% for 16-carbon chain lipid membranes, and >40% for 18-chain lipid membranes, which was surprising as 25% is used clinically in LiPlaCis®. Efficient sPLA2-dependent growth inhibition of colorectal cancer cells was demonstrated in vitro, where cell membrane degradation and cytolysis depends on the sensitivity of the formulation towards the enzyme and is governed by the amount of lysolipids generated and the presence of serum proteins. We found that serum proteins did not affect the lipase activity of the enzyme towards the membranes but instead sequester the lysolipid byproducts consequently inhibiting their detergent-like cytotoxic properties. In vivo therapeutic potential and safety of the liposomes was investigated in nude mice bearing sPLA2-deficient FaDu squamous carcinoma and sPLA2-expressing Colo205 colorectal adenocarcinoma. After intravenous injections, the tumor growth was suppressed for liposomal L-OHP relative to free drug, but only a weak response was observed for both slow- and fast-releasing sPLA2-sensitive formulations compared to non-sensitive liposomes. Also, the mice did not show longer survival. In turn, for the highly sPLA2-sensitive liposomes, multiple high doses caused petechial cutaneous hemorrhages, along with multifocal hepatonecrotic lesions, suggestive of premature activation in skin and liver irrespective of sPLA2-status of the tumor engraft. These results indicate that although liposomal carriers can improve the antitumor efficacy of platinum drugs, sPLA2-triggered release suffers from a narrow therapeutic index and has safety concerns.

Aptatope mapping of the binding site of a progesterone aptamer on the steroid ring structure.

In this work we report the mapping of the binding site of the only progesterone aptamer published to date, in an approach referred to as aptatope mapping. By linking the binding data obtained from microscale thermophoresis analysis to the structural differences on the ring structure of a range of steroids, we elucidated the moieties involved in aptamer-progesterone binding. This approach can be further exploited for the characterization of aptamer specificity and ultimately facilitate the development of aptamer-based assays depending on the desired specificity.

Electrochemical Genetic Profiling of Single Cancer Cells.

Recent understandings in the development and spread of cancer have led to the realization of novel single cell analysis platforms focused on circulating tumor cells (CTCs). A simple, rapid, and inexpensive analytical platform capable of providing genetic information on these rare cells is highly desirable to support clinicians and researchers alike to either support the selection or adjustment of therapy or provide fundamental insights into cell function and cancer progression mechanisms. We report on the genetic profiling of single cancer cells, exploiting a combination of multiplex ligation-dependent probe amplification (MLPA) and electrochemical detection. Cells were isolated using laser capture and lysed, and the mRNA was extracted and transcribed into DNA. Seven markers were amplified by MLPA, which allows for the simultaneous amplification of multiple targets with a single primer pair, using MLPA probes containing unique barcode sequences. Capture probes complementary to each of these barcode sequences were immobilized on a printed circuit board (PCB) manufactured electrode array and exposed to single-stranded MLPA products and subsequently to a single stranded DNA reporter probe bearing a HRP molecule, followed by substrate addition and fast electrochemical pulse amperometric detection. We present a simple, rapid, flexible, and inexpensive approach for the simultaneous quantification of multiple breast cancer related mRNA markers, with single tumor cell sensitivity.

The characterization and validation of 17ß-estradiol binding aptamers.

The rapid and sensitive detection of small molecules is garnering increasing importance, and aptamers show great promise in replacing expensive, elaborate detection platforms exploiting chromatographic separation or antibody-based assays. The characterization of aptamer interaction with small molecule targets is not facile, and there is a mature need for a rapid, high-throughput technique for the analysis of aptamer-small molecule kinetics and affinity. In this work we present methodologies for the evaluation of aptamer-small molecule interactions, using the aptamers reported against the steroid 17ß-estradiol as a model system. Microscale thermophoresis, apta-PCR affinity assay and surface plasmon resonance were explored to evaluate the reported aptamers' binding properties in terms of affinity and specificity, and were demonstrated to be successfully applied to the analysis of aptamer-small molecule interactions.

Sensitive detection of cancer cells using light-mediated apta-PCR.

Apta-PCR is an ultrasensitive assay in which aptamers are exploited not only as biomolecular recognition elements, but also as reporter labels for amplification via real-time PCR. This methodology has been successfully applied to the detection of proteins, achieving limits of detection in the picomolar range. The introduction of caged aptamers that bear photo-labile groups, so called cages, at strategic positions so that their tertiary structure and thus their binding properties can be controlled by light, facilitates a more robust and attractive assay in terms of sample conservation and reusability. In this work, we report for the first time the use of caged aptamers for cell detection in an apta-PCR assay. Specifically, a sandwich format is used combining the capture of B-cells by an antibody with the specific detection of Burkitt's lymphoma cancer cells by a caged aptamer, acting as a reporter probe. Elution of the aptamer bound to the cancer cells is performed by light and the number of cells is then correlated with the amount of eluted caged aptamer using real-time PCR analysis. The reported technique shows an excellent sensitivity, achieving detection of as few as 77 cells, and due to the inherent robustness of the assay, this detection platform can be reused for further analyses, demonstrating potential applicability in proteomics and clinical diagnostics.

A simple liposome assay for the screening of zinc ionophore activity of polyphenols.

An efficient liposomal system for screening the zinc ionophore activity of a selected library consisting of the most relevant dietary polyphenols is presented. The zinc ionophore activity was demonstrated by exploring the use of zinc-specific fluorophore FluoZin-3 loaded liposomes as simple membrane tools that mimic the cell membrane. The zinc ionophore activity was demonstrated as the capacity of polyphenols to transport zinc cations across the liposome membrane and increase the zinc-specific fluorescence of the encapsulated fluorophore FluoZin-3. In addition, the zinc chelation strength of the polyphenols was also tested in a competition assay based on the fluorescence quenching of zinc-dependent fluorescence emitted by zinc-FluoZin-3 complex. Finally, the correlation between the chelation capacity and ionophore activity is demonstrated, thus underlining the sequestering or ionophoric activity that the phenolic compounds can display, thus, providing better knowledge of the importance of the structural conformation versus their biological activity. Furthermore, the assays developed can be used as tools for rapid, high-throughput screening of families of polyphenols towards different biometals.

New platforms for multi-functional ocular lenses: engineering double-sided functionalized nano-coatings.

A scalable platform to prepare multi-functional ocular lenses is demonstrated. Using rapidly dissolving polyvinylpyrrolidone (PVP) as the active stabilizing matrix, both sides of ocular lenses were coated using a modified scaled-up masking electrohydrodynamic atomization (EHDA) technique (flow rates variable between 5 and 10 µL/min, applied voltage 4-11 kV). Each side was coated (using a specially designed flip-able well) selectively with a pre-determined morphology and model drug substance. PVP nanoparticles (inner side, to be in contact with the cornea, mean size

Zinc ionophore activity of quercetin and epigallocatechin-gallate: from Hepa 1-6 cells to a liposome model.

Labile zinc, a tiny fraction of total intracellular zinc that is loosely bound to proteins and easily interchangeable, modulates the activity of numerous signaling and metabolic pathways. Dietary plant polyphenols such as the flavonoids quercetin (QCT) and epigallocatechin-gallate act as antioxidants and as signaling molecules. Remarkably, the activities of numerous enzymes that are targeted by polyphenols are dependent on zinc. We have previously shown that these polyphenols chelate zinc cations and hypothesized that these flavonoids might be also acting as zinc ionophores, transporting zinc cations through the plasma membrane. To prove this hypothesis, herein, we have demonstrated the capacity of QCT and epigallocatechin-gallate to rapidly increase labile zinc in mouse hepatocarcinoma Hepa 1-6 cells as well as, for the first time, in liposomes. In order to confirm that the polyphenols transport zinc cations across the plasma membrane independently of plasma membrane zinc transporters, QCT, epigallocatechin-gallate, or clioquinol (CQ), alone and combined with zinc, were added to unilamellar dipalmitoylphosphocholine/cholesterol liposomes loaded with membrane-impermeant FluoZin-3. Only the combinations of the chelators with zinc triggered a rapid increase of FluoZin-3 fluorescence within the liposomes, thus demonstrating the ionophore action of QCT, epigallocatechin-gallate, and CQ on lipid membrane systems. The ionophore activity of dietary polyphenols may underlay the raising of labile zinc levels triggered in cells by polyphenols and thus many of their biological actions.

Evaluation of techniques for generation of single-stranded DNA for quantitative detection.

A simple and efficient method for the generation of clean single-stranded DNA (ssDNA) with a high recovery and purity from a double-stranded polymerase chain reaction (PCR) product is required for nucleic acid sensing and microarray applications. Currently, the most widely used technique is thermal denaturation due to its simplicity and low cost, but this technique has drawbacks in terms of recovery and reproducibility. The work presented here compares this technique with alternative approaches for ssDNA generation exploiting affinity magnetic separation and exonuclease digestion. The ssDNA recovered was evaluated using gel electrophoresis and enzyme-linked oligonucleotide assay. Recoveries of between 50% and 70% of the theoretical maximum of generatable ssDNA were obtained with good reproducibility, demonstrating a marked improvement in performance as compared with thermal denaturation.

Electrochemical genosensor array for the simultaneous detection of multiple high-risk human papillomavirus sequences in clinical samples.

An electrochemical genosensor array for the simultaneous detection of three high-risk human papillomavirus (HPV) DNA sequences, HPV16, 18 and 45, exhibiting high sensitivity and selectivity is presented. The electrodes of a 4×4 array were modified via co-immobilization of a 1:100 (mol/mol) mixture of a thiolated probe and an oligoethyleneglycol-terminated bipodal thiol. Detection of synthetic and PCR products was carried out in a sandwich type format, with the target hybridized between a surface immobilized probe and a horseradish peroxidase-labelled secondary reporter probe. The detection limits obtained in the detection of each individual target were in the pM range, allowing the application of this sensor for the detection of samples obtained from PCR amplification of cervical scrape samples. The results obtained exhibited an excellent correlation with the HPV genotyping carried out within a hospital laboratory. Multiplexing and cross-reactivity studies demonstrated high selectivity over potential interfering sequences, facilitating application of the developed platform for the high-throughput screening of multiple high-risk DNA sequences.

Three-dimensional arrangement of short DNA oligonucleotides at surfaces via the synthesis of DNA-branched polyacrylamide brushes by SI-ATRP.

Short DNA oligonucleotide branches are incorporated into acrylamide brushes via surface initiated atom transfer radical polymerization in an attempt to increase DNA surface density by building three-dimensional molecular architectures. ATR-FTIR as well as hybridization studies followed by SPR confirm the incorporation of the DNA sequences into the polymer backbone. MALDI-TOF analysis further suggests that six acrylamide monomer units are typically separating DNA branches present on a single brushes approximately 26 units long. This new approach offers a promising alternative to SAM-based nucleic acid and aptamer sensors and could enable the realization of more complex soft materials of controlled architecture capable of both recognition and signaling by including additional optically or electrochemically active moieties.

Electrochemical detection of celiac disease-related anti-tissue transglutaminase antibodies using thiol based surface chemistry.

Celiac disease is an autoimmune disorder that affects the gastrointestinal tract upon ingestion of gluten, which triggers the production of antibodies against gliadin and tissue transglutaminase, activating an inflammatory response and inducing tissue damage in the small intestine resulting in malabsorption. The measurement of these antibodies in an individual's blood can be used to screen for celiac disease and the criteria for definitive diagnosis is currently being revised to be based on serological analysis rather than biopsy. In the work reported here, an electrochemical immunosensor for the detection of human anti-tissue transglutaminase antibodies was developed, consisting of gold-based self-assembled monolayers of a carboxylic group terminated bipodal alkanethiol that is covalently linked to tissue transglutaminase, the antigen for the immunorecognition of circulating autoantibodies. The presence of the autoantibodies was recorded using horseradish peroxidase labeled anti-human antibodies, which provided an enzyme based electrochemical signal. Optimization and characterization of the surface of the sensor was carried out by electrochemical impedance spectroscopy and surface plasmon resonance. The immunosensor gave a stable quantitative response to different antibody concentrations after 30 min with a limit of detection of 390 ng/mL and an RSD of 9%, n=3. The developed immunosensor was tested with calibrator solutions as well as with real patients' samples, and the results compared to those obtained from Eurospital's Eu-tTG IgA and IgG ELISA kits, showing an excellent degree of correlation.

Automated microsystem for electrochemical detection of cancer markers.

The development of a fully automated microsystem housing an amperometric immunosensor is presented. The microfluidic cell integrates reagent storage and electrochemical immunodetection and was applied for the detection of breast cancer markers. The main advantage of this system is that no external fluidic storage is required and the instrumental setup is thus greatly simplified. The fluidics of the microsystem is computer controlled and requires minimal end-user intervention. The analytical performance of the device was compared with a manually driven system and applied for the amperometric detection of the carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA15-3). This automation methodology greatly improves the analytical performance of the immunosensor in terms of accuracy and reproducibility as evidenced by a reduction of LOD observed for CEA and CA15-3 with respect to a manually driven system. Finally, the automated microsystem was applied for the analysis of real patient serum samples, demonstrating excellent correlation with a commercial ELISA.

Integrated microfluidic platform for the electrochemical detection of breast cancer markers in patient serum samples.

A microsystem integrating electrochemical detection for the simultaneous detection of protein markers of breast cancer is reported. The microfluidic platform was realized by high precision milling of polycarbonate sheets and features two well distinguishable sections: a detection zone incorporating the electrode arrays and the fluid storage part. The detection area is divided into separate microfluidic chambers addressing selected electrodes for the measurement of samples and calibrators. The fluidic storage part of the platform consists of five reservoirs to store the reagents and sample, which are interfaced by septa. These reservoirs have the appropriate volume to run a single assay per cartridge and are manually filled. The liquids from the reservoirs are actuated by applying a positive air pressure (i.e.via a programmable syringe pump) through the septa and are driven to the detection zone via two turning valves. The application of the realised platform in the individual and simultaneous electrochemical detection of proteic cancer markers with very low detection limits are demonstrated. The microsystem has also been validated using real patient serum samples and excellent correlation with ELISA results obtained.

Signal-enhancing thermosensitive liposomes for highly sensitive immunosensor development.

Liposomes are potential candidates as nanovesicles for the development of detection systems with improved sensitivity and detection limits, due to their capacity to encapsulate diverse types of signal enhancing molecules. An amperometric immunosensor exploiting enzyme encapsulating thermosensitive liposomes for the ultrasensitive detection of carcinoembryonic antigen (CEA) is reported. Five different bioconjugation methods to link an anti-CEA antibody to horseradish peroxidase (HRP) encapsulating liposomes were studied and compared to HRP-Ab conjugate. ζ-Potential measurements of liposomes before and after each modification method as well as following incubation with CEA were used as a tool to monitor the success of modification and probe the affinity of the liposome linked antibodies. The use of different lysing conditions (temperature vs detergent) was evaluated, with the application of temperature providing an extremely effective means of liposome lysis. Finally, thermosensitive liposomes modified using biotin-streptavidin and N-succinimidyl-S-acetylthioacetate (SATA)/sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-1-carboxylate (Sulfo-SMCC) chemistries were used to detect CEA and compared in terms of their stability, background signal, and limit of detection. Detection limits of 2 orders of magnitude lower than that obtained with the HRP-antibody reporter conjugate were obtained (0.080 ng CEA/mL and 0.0113 ng CEA/mL), with 11-fold and 9-fold amplification of signal, for the biotin-streptavidin and SATA/Sulfo-SMCC modified liposomes respectively, clearly demonstrating the powerful potential of enzyme encapsulating liposomes as signal enhancement tools.

Lab-on-a-chip for the isolation and characterization of circulating tumor cells.

A smart miniaturized system is being proposed for the isolation and characterization of circulating tumor cells (CTCs) directly from blood. Different microfluidic modules have been designed for cell enrichment and -counting, multiplex mRNA amplification as well as DNA detection. With the different modules at hand, future effort will focus on the integration of the modules in a fully automated, single platform.

Automated genotyping of circulating tumor cells.

Cancer remains a prominent health concern in modern societies. Continuous innovations and introduction of new technologies are essential to level or reduce current healthcare spending. A diagnostic platform to detect circulating tumor cells (CTCs) in peripheral blood may be most promising in this respect. CTCs have been proposed as a minimally invasive, prognostic and predictive marker to reflect the biological characteristics of tumors and are implemented in an increasing number of clinical studies. Still, their detection remains a challenge as they may occur at concentrations below one single cell per ml of blood. To facilitate their detection, here we describe microfluidic modules to isolate and genotype CTCs directly from clinical blood samples. In a first cell isolation and detection module, the CTCs are immunomagnetically enriched, separated and counted. In a second module and after cell lysis, the mRNA is reversely transcripted to cDNA, followed by a multiplex ligation probe amplification of 20 specific genetic markers and two control fragments. Following the multiplex ligation probe amplification reaction, the amplified fragments are electrochemically detected in a third and final module. Besides the design of the modules, their functionality is described using control samples. Further testing using clinical samples and integration of all modules in a single, fully automated smart miniaturized system will enable minimal invasive testing for frequent detection and characterization of CTCs.

Development of a gold nano-particle-based fluorescent molecular beacon for detection of cystic fibrosis associated mutation.

Cystic fibrosis is one of the most common genetically inherited diseases in Northern Europe, consisting of an inherited defect of chloride transport in the epithelium. Of the several mutations related to CF, the ΔF508 mutation occurs in ca. 70% of the cases. In this work the use of a gold nano-particle supported fluorescence molecular beacon was investigated as an optical sensing platform for the detection of the ΔF508 cystic fibrosis associated mutation. Different parameters such as molecular beacon design, Au nano-particle size, molecular beacon-nano-particle conjugation protocol, molecular beacon loading as well as experimental conditions were evaluated. A 31-base long molecular beacon, containing a 15-base recognition sequence specific for the mutant target, was linked via a thiol modified poly thymine linker (10 bases long) to a 13 nm gold nano-particle and was exposed to mutant and wild type targets, and a clear differentiation was achieved at target concentrations as low as 1 nM.