Relationship between the Expression of Matrix Metalloproteinases and Their Tissue Inhibitors in Patients with Brain Tumors.

Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) play critical roles in regulating processes associated with malignant behavior. These endopeptidases selectively degrade components of the extracellular matrix (ECM), growth factors, and their receptors, contributing to cancer cell invasiveness and migratory characteristics by disrupting the basal membrane. However, the expression profile and role of various matrix metalloproteinases remain unclear, and only a few studies have focused on differences between diagnoses of brain tumors. Using quantitative real-time PCR analysis, we identified the expression pattern of ECM modulators (n = 10) in biopsies from glioblastoma (GBM; n = 20), astrocytoma (AST; n = 9), and meningioma (MNG; n = 19) patients. We found eight deregulated genes in the glioblastoma group compared to the benign meningioma group, with only MMP9 (FC = 2.55; p = 0.09) and TIMP4 (7.28; p < 0.0001) upregulated in an aggressive form. The most substantial positive change in fold regulation for all tumors was detected in matrix metalloproteinase 2 (MNG = 30.9, AST = 4.28, and GBM = 4.12). Notably, we observed an influence of TIMP1, demonstrating a positive correlation with MMP8, MMP9, and MMP10 in tumor samples. Subsequently, we examined the protein levels of the investigated MMPs (n = 7) and TIMPs (n = 3) via immunodetection. We confirmed elevated levels of MMPs and TIMPs in GBM patients compared to meningiomas and astrocytomas. Even when correlating glioblastomas versus astrocytomas, we showed a significantly increased level of MMP1, MMP3, MMP13, and TIMP1. The identified metalloproteases may play a key role in the process of gliomagenesis and may represent potential targets for personalized therapy. However, as we have not confirmed the relationship between mRNA expression and protein levels in individual samples, it is therefore natural that the regulation of metalloproteases will be subject to several factors.

Use of rapid diagnostic tests for the detection of ancient malaria infections in dental pulp from the sixth century in Versailles, France.

BACKGROUND: Paleomicrobiological data have clarified that Plasmodium spp. was circulating in the past in southern European populations, which are now devoid of malaria. The aim of this study was to evaluate the efficacy of immunodetection and, more particularly, rapid diagnostic tests (RDT), in order to further assess Plasmodium infections in ancient northern European populations. METHODS: A commercially available RDT, PALUTOP® + 4 OPTIMA, which is routinely used to detect malaria, was used to detect Plasmodium antigens from proteins recovered from ancient specimens extracted from 39 dental pulp samples. These samples were collected from 39 individuals who were buried in the sixth century, near the site of the current Palace of Versailles in France. Positive and negative controls were also used. Antigens detected were quantified using chemiluminescence imaging system analysis. RESULTS: Plasmodium antigens were detected in 14/39 (35.9%) individuals, including Plasmodium vivax antigens in 11 individuals and Plasmodium falciparum antigens co-detected in two individuals, while Pan-Plasmodium antigens were detected in three individuals. Controls all yielded expected results. CONCLUSIONS: The data reported here showed that RDTs are a suitable tool for detecting Plasmodium spp. antigens in ancient dental pulp samples, and demonstrated the existence of malaria in Versailles, France, in the sixth century. Plasmodium vivax, which is regarded as being responsible for an attenuated form of malaria and less deadly forms, was the most prevalent species. This illustrates, for the first time in ancient populations, co-infection with P. falciparum, bringing into question the climate-driven ecosystems prevailing at that time in the Versailles area.

Highly sensitive digital detection of SARS-CoV-2 nucleocapsid protein through single-molecule counting.

Nucleocapsid protein (NP) is one of the structural proteins of SARS-CoV-2 which is stable, well-conserved, highly immunogenic, and abundantly expressed due to the host's adaptive immune response, making it a promising antigenic biomarker for the early and rapid identification and diagnosis of SARS-CoV-2. Traditional antigen analytical methods with NP as the detection marker often have insufficient sensitivity. To achieve rapid and highly sensitive detection of NP, we constructed a novel single-molecule (digital) fluorescence-linked immunosorbent assay (FLISA) based on streptavidin-modified transparent 96-well microplates. Streptavidin was immobilized on the microplate under optimized conditions with a 15 mM carbonate buffer solution (pH 9.6) as the coating solution, biotinylated antibodies conjugated with streptavidin as capture probes, and carboxylated fluorescent microsphere-conjugated monoclonal antibodies (FMs-mAbs) as fluorescent probes. Individual sandwich immunolabeled complexes of the SARS-CoV-2 diagnostic marker NP were detected and counted though wide-field inverted fluorescence microscopy (1.1 × 1.4 mm2). FLISA had a linear detection range of 0.2 pg/mL to 200 ng/mL and a limit of detection (LOD) of 0.73 fg/mL and 8 fg/mL for NP in phosphate buffer saline and spiked nasal swab samples, respectively. The sensitivity was much higher than commercial antigen detection kits, providing wide detection prospects in future clinical diagnosis, environmental monitoring, and other fields.

Triangular Silver Nanoplates as a Bioanalytical Tool: Potential COVID-19 Detection.

Nanotechnology offers new possibilities in molecular diagnostics, with nanoparticles gaining attention as biosensor upgrades. This study evaluates gold-coated silver nanoplates coated with PEG for enhanced protection, aiming to detect Spike protein with higher sensitivity, and emphasizes the importance of considering complex environments and appropriate controls for specific binding and accurate analysis. The sensitivity of antibody-coated PEGAuTSNPs as tools for immunoassays is demonstrated through fibronectin (Fn)- anti-fibronectin binding within an isolated extracellular matrix as a complex and native environment of Fn. Moreover, the optimal functionalization volume of Spike protein was determined (4 µg/mL of PEGAuTSNP). Anti-Spike was added to confirm binding, while the TJP1 protein was used as a negative control. The same experiment was used in the presence of horse serum to simulate a complex environment. According to Localized Surface Plasmon Resonance analysis and Dynamic Light Scattering size measurements, anti-Spike exhibited a stronger affinity for the nanoplates, causing TJP1 to be replaced by the antibody on the nanoplates' surface. Future research will involve exploring alternative complex environments, filtering larger molecules, and the optimization of immunoassay performance.

Screening of monoclonal antibodies against specific phosphonylation sites and analysis of serum samples exposed to soman and VX using an indirect competitive enzyme-linked immunosorbent assay.

Organophosphorus nerve agents (OPNAs) covalently bind to tyrosine 411 of human serum albumin (HSA) and the formed adducts are stable biomarkers of OPNA exposure. The detection of these adducts has been limited to mass spectrometry techniques combined with protein digestion. Here, we developed indirect competitive ELISA (icELISA) methods to verify OPNA exposure by the detection of OPNA-phosphonylated adducts at tyrosine 411 residue (OPNA-HSA adducts), in which monoclonal antibodies (mAbs) against phosphonylation sites at tyrosine 411 were introduced. The two mAbs were prepared by the fourth generation of rabbit mAb technology using the phosphonylated peptides of LVRY(GD or VX)TKKVPQC as the haptens. These mAbs were screened using our developed competitive ELISA method and then selected based on their individual affinity and selectivity. As a result, we obtained two mAbs that recognized the HSA Tyr 411 adduct of GD (mAb-5G2) or VX (mAb-12B9), respectively. They shared the highest affinity exhibiting a Kd value of about 10-6 mol/L of the OPNA exposure concentration. They also had remarkable selectivity, which could especially recognize their individual OPNA-HSA adducts in a native state but did not recognize other OPNA-HSAs and unadducted HSAs. Especially for mAb-12B9, it could clearly distinguish VX-HSA and GB-HSA between which there was only one alkyl difference in their phosphonyl portion of the adducted sites. The two mAbs were then used to build the icELISA method for analysis of the serum samples exposed to OPNA. It was found that the detectable lowest GD- and VX-exposed concentrations in serum samples were respectively 1.0 × 10-6 mol/L and 10.0 × 10-6 mol/L. This study provides one novel approach and strategy for the retrospective detection of OPNA exposure, and the two mAbs have great potential to be extended for point-of-care testing of OPNA intoxication.

Kinetic Analysis of Prostate-Specific Antigen Interaction with Monoclonal Antibodies for Development of a Magnetic Immunoassay Based on Nontransparent Fiber Structures.

Prostate cancer is the second most common cancer diagnosed in men worldwide. Measuring the prostate-specific antigen (PSA) is regarded as essential during prostate cancer screening. Early diagnosis of this disease relapse after radical prostatectomy requires extremely sensitive methods. This research presents an approach to development of an ultrasensitive magnetic sandwich immunoassay, which demonstrates the limit of PSA detection in human serum of 19 pg/mL at a dynamic range exceeding 3.5 orders of concentration. Such attractive performance stems, inter alia, from the kinetic analysis of monoclonal antibodies (mAbs) against free PSA to select the mAbs exhibiting best kinetic characteristics and specificity. The analysis is carried out with a label-free multiplex spectral-correlation interferometry compatible with inexpensive single-use glass sensor chips. The high sensitivity of developed PSA immunoassay is due to electronic quantification of magnetic nanolabels functionalized by the selected mAbs and three-dimension porous filters used as an extended solid phase. The assay is promising for PSA monitoring after radical prostatectomy. The proposed versatile approach can be applied for the rational design of highly sensitive tests for detection of other analytes in many fields, including in vitro diagnostics, veterinary, food safety, etc.

Molecular tools for the characterization of seizure susceptibility in genetic rodent models of epilepsy.

Epilepsy is a chronic neurological disorder characterized by abnormal neuronal activity that arises from imbalances between excitatory and inhibitory synapses, which are highly correlated to functional and structural changes in specific brain regions. The difference between the normal and the epileptic brain may harbor genetic alterations, gene expression changes, and/or protein alterations in the epileptogenic nucleus. It is becoming increasingly clear that such differences contribute to the development of distinct epilepsy phenotypes. The current major challenges in epilepsy research include understanding the disease progression and clarifying epilepsy classifications by searching for novel molecular biomarkers. Thus, the application of molecular techniques to carry out comprehensive studies at deoxyribonucleic acid, messenger ribonucleic acid, and protein levels is of utmost importance to elucidate molecular dysregulations in the epileptic brain. The present review focused on the great diversity of technical approaches available and new research methodology, which are already being used to study molecular alterations underlying epilepsy. We have grouped the different techniques according to each step in the flow of information from DNA to RNA to proteins, and illustrated with specific examples in animal models of epilepsy, some of which are our own. Separately and collectively, the genomic and proteomic techniques, each with its own strengths and limitations, provide valuable information on molecular mechanisms underlying seizure susceptibility and regulation of neuronal excitability. Determining the molecular differences between genetic rodent models of epilepsy and their wild-type counterparts might be a key in determining mechanisms of seizure susceptibility and epileptogenesis as well as the discovery and development of novel antiepileptic agents. This article is part of the Special Issue "NEWroscience 2018".

Brain Protein Expression Profile Confirms the Protective Effect of the ACTH(4-7)PGP Peptide (Semax) in a Rat Model of Cerebral Ischemia-Reperfusion.

The Semax (Met-Glu-His-Phe-Pro-Gly-Pro) peptide is a synthetic melanocortin derivative that is used in the treatment of ischemic stroke. Previously, studies of the molecular mechanisms underlying the actions of Semax using models of cerebral ischemia in rats showed that the peptide enhanced the transcription of neurotrophins and their receptors and modulated the expression of genes involved in the immune response. A genome-wide RNA-Seq analysis revealed that, in the rat transient middle cerebral artery occlusion (tMCAO) model, Semax suppressed the expression of inflammatory genes and activated the expression of neurotransmitter genes. Here, we aimed to evaluate the effect of Semax in this model via the brain expression profiling of key proteins involved in inflammation and cell death processes (MMP-9, c-Fos, and JNK), as well as neuroprotection and recovery (CREB) in stroke. At 24 h after tMCAO, we observed the upregulation of active CREB in subcortical structures, including the focus of the ischemic damage; downregulation of MMP-9 and c-Fos in the adjacent frontoparietal cortex; and downregulation of active JNK in both tissues under the action of Semax. Moreover, a regulatory network was constructed. In conclusion, the suppression of inflammatory and cell death processes and the activation of recovery may contribute to the neuroprotective action of Semax at both the transcriptome and protein levels.

Improvement of immunodetection of the transcription factor C/EBP homologous protein by western blot.

Accumulation of misfolded proteins within the endoplasmic reticulum (ER) induces an unfolded protein response (UPR) that either restores homeostasis or triggers apoptosis in case of adaptation failure. The three activated branches of UPR lead to IRE1-, PERK- and ATF6- dependent transcriptional induction of the gene encoding the transcription factor C/EBP homologous protein (CHOP) which plays an important role in apoptosis induction. In conventional immunoblotting conditions, detection of CHOP is a difficult task. Using a fixation step, we have optimized the detection of CHOP and this method provides a valuable tool to decipher CHOP involvement in UPR.

Detection of fly artifacts from four species of necrophagous flies on household materials using immunoassays.

An immunoassay was previously developed as a technique to improve methods for detection and analysis of fly artifacts found at crime scenes. The dot blot assay utilized a polyclonal antiserum (anti-md3) based on a unique digestive cathepsin D found in cyclorrhaphous Diptera. In this study, artifacts produced by adults of Calliphora vicina, Cynomya cadaverina, Sarcophaga bullata, and Protophormia terraenovae were examined using the immunoassay to determine if insect-derived stains could be distinguished from a range of human body fluid stains. A lift technique was developed which permitted transfer of fly artifacts from test materials to filter paper for dot blot analyses. All species readily deposited artifacts on all test household materials regardless of diet consumed. Despite differences in texture and porosity of the household materials, artifacts of all species transferred to the filter paper. With all fly species, anti-md3 serum bound to artifacts produced after feeding on semen, blood, feces, urine, and saliva. By contrast, anti-md3 serum did not react with any of the human fluids tested, nor with any of the lifts from household materials not exposed to flies. There was no evidence of false positives with any of the fly species tested, regardless of diet consumed. There was also no indication of false negatives with any of the dot blot assays. These observations suggest that immunoassays using anti-md3 serum performed on a simple lift of suspected fly artifacts can be used effectively as a confirmatory assay to distinguish fly regurgitate and fecal stains from human body fluids.

High-performance detection of an abdominal aortic aneurysm biomarker by immunosensing.

Abdominal aortic aneurysm (AAA) is a serious, life-threatening vascular disease that presents as an enlarged area of the aorta, which is the main artery that carries blood away from the heart. AAA may occur at any location in the aorta, but it is mainly found in the abdominal region. A ruptured AAA causes serious health issues, including death. Traditional imaging techniques, such as computed tomography angiogram, magnetic resonance imaging, and ultrasound sonography, have been used to identify AAAs. Circulating biomarkers have recently become attractive for diagnosing AAAs due to their cost-effectiveness compared to imaging. Insulin-like growth factor 1 (IGF-1), a secreted hormone vital for human atherosclerotic plaque stability, has been found to be an efficient biomarker for AAA identification. In this report, immunosensing was performed by using an InterDigitated electrode (IDE) sensor to detect circulating levels of IGF-1. The detection limit of IGF-1 was found to be 100 fM with this sensor. Moreover, related protein controls (IGF-2 and IGFBP3) were not detected with the same antibody, indicating selective IGF-1 detection. Thus, immunosensing by using an IDE sensor may help to effectively diagnose AAAs and represents a basic platform for further development.

Sensor Based on PZT Ceramic Resonator with Lateral Electric Field for Immunodetectionof Bacteria in the Conducting Aquatic Environment.

A biological sensor for detection and identification of bacterial cells, including a resonator with a lateral electric field based on PZT ceramics was experimentally investigated. For bacterial immunodetection the frequency dependencies of the electric impedance of the sensor with a suspension of microbial cells were measured before and after adding the specific antibodies. It was found that the addition of specific antibodies to a suspension of microbial cells led to a significant change in these frequency dependencies due to the increase in the conductivity of suspension. The analysis of microbial cells was carried out in aqueous solutions with a conductivity of 4.5-1000 µS/cm, as well as in the tap and drinking water. The detection limit of microbial cells was found to be 103 cells/mLand the analysis time did not exceed 4 min. Experiments with non-specific antibodies were also carried out and it was shown that their addition to the cell suspension did not lead to a change in the analytical signal of the sensor. This confirms the ability to not only detect, but also identify bacterial cells in suspensions.

Optomechanical devices for deep plasma cancer proteomics.

Most of the cancer deaths could be avoided by early detection of the tumor when it is confined to its primary site and it has not metastasized. To this aim, one of the most promising strategies is the discovery and detection of protein biomarkers shed by the young tumor to the bloodstream. Proteomic technologies, mainly mass spectrometry and multiplexed immunoassays, have rapidly developed during last years with improved limits of detection and multiplexing capability. Unfortunately, these developments together major investments and large international efforts have not resulted into new useful protein biomarkers. Here, we analyze the potential and limitations of current proteomic technologies for detecting protein biomarkers released into circulation by the tumor. We find that these technologies can hardly probe the deepest region of the plasma proteome, at concentrations below the pg/mL level, where protein biomarkers for early cancer detection may exist. This clearly indicates the need of incorporating novel ultrasensitive techniques to the proteomic tool-box that can cover the inaccessible regions of the plasma proteome. We here propose biological detectors based on nanomechanical systems for discovery and detection of cancer protein biomarkers in plasma. We review the modes of operation of these devices, putting our focus on recent developments on nanomechanical sandwich immunoassays and nanomechanical spectrometry. The first technique enables reproducible immunodetection of proteins at concentrations well below the pg/mL level, with a limit of detection on the verge of 10 ag/mL. This technology can potentially detect low abundance tumor-associated proteins in plasma at the very early stages of the tumor. The second technique enables the identification of individual intact proteins by two physical coordinates, the mass and stiffness, instead of the mass-to-charge ratio of the protein constituents. This technology enormously simplifies the identification of proteins and it can provide useful information on interactions and posttranslational modifications, that otherwise is lost in mass spectrometry.

Whole-bacterium ribosome display selection for isolation of highly specific anti-Staphyloccocus aureus Affitins for detection- and capture-based biomedical applications.

Detection and capture methods using antibodies have been developed to ensure identification of pathogens in biological samples. Though antibodies have many attractive properties, they also have limitations and there are needs to expand the panel of available affinity proteins with different properties. Affitins, that we developed from the Sul7d proteins, are a solid class of affinity proteins, which can be used as substitutes to antibodies or to complement them. We report the generation and characterization of antibacterial Affitins with high specificity for Staphylococcus aureus. For the first time, ribosome display selections were carried out using whole-living-cell and naïve combinatorial libraries, which avoid production of protein targets and immunization of animals. We showed that Affitin C5 exclusively recognizes S. aureus among dozens of strains, including clinical ones. C5 binds staphylococcal Protein A (SpA) with a K D of 108 ± 2 nM and has a high thermostability (T m = 77.0°C). Anti-S. aureus C5 binds SpA or bacteria in various detection and capture applications, including ELISA, western blot analysis, bead-fishing, and fluorescence imaging. Thus, novel anti-bacteria Affitins which are cost-effective, stable, and small can be rapidly and fully designed in vitro with high affinity and specificity for a surface-exposed marker. This class of reagents can be useful in diagnostic and biomedical applications.

An origami paper-based electrochemical immunoassay for the C-reactive protein using a screen-printed carbon electrode modified with graphene and gold nanoparticles.

An origami paper-based electrochemical immunoassay for C-reactive protein (CRP) detection is described. The assay integrates multiple steps of electrode modification into a single device. A graphene-modified screen-printed carbon electrode (G/SPCE) was employed to enhance sensitivity. Gold nanoparticles were first electrodeposited onto the G/SPCE, followed by a self-assembled monolayer of L-cysteine. The capture anti-CRP was then covalently immobilized on the modified electrode. CRP was quantified by measuring the changes in the charge-transfer resistance of the electrode by using hexacyanoferrate as the redox probe. Cyclic voltammetry and scanning electron microscopy were also applied to verify the successful modification of the electrode. Under optimal conditions, impedance increase in the 0.05-100 µg mL-1 CRP concentration range, and the limit of detection is 15 ng mL-1 (at S/N = 3). The immunoassay was successfully applied to the determination of CRP in a certified human serum sample. This method is simple, low-cost, portable and disposable. Graphical abstract An origami paper-based analytical device (oPAD) is described that integrates the multistep of electrode modification, immobilization and detection into a single device. The direct conjugation between the capture antibody and target molecule was allowed to use in this system. The C-reactive protein (CRP) concentration in serum samples was determined using electrochemical impedance spectroscopy.

Simultaneous Immunodetection of Anthrax, Plague, and Tularemia from Blood Cultures by Use of Multiplexed Suspension Arrays.

Multiplexed detection technologies are becoming increasingly important given the possibility of bioterrorism attacks, for which the range of suspected pathogens can vary considerably. In this work, we describe the use of Luminex MagPlex magnetic microspheres for the construction of two multiplexed diagnostic suspension arrays, enabling antibody-based detection of bacterial pathogens and their related disease biomarkers directly from blood cultures. The first 4-plex diagnostic array enabled the detection of both anthrax and plague infections using soluble disease biomarkers, including protective antigen (PA) and anthrax capsular antigen for anthrax detection and the capsular F1 and LcrV antigens for plague detection. The limits of detection (LODs) ranged between 0.5 and 5 ng/ml for the different antigens. The second 2-plex diagnostic array facilitated the detection of Yersinia pestis (LOD of 1 × 106 CFU/ml) and Francisella tularensis (LOD of 1 × 104 CFU/ml) from blood cultures. Inoculated, propagated blood cultures were processed (15 to 20 min) via 2 possible methodologies (Vacutainer or a simple centrifugation step), allowing the direct detection of bacteria in each sample, and the entire assay could be performed in 90 min. While detection of bacteria and soluble markers from blood cultures using PCR Luminex suspension arrays has been widely described, to our knowledge, this study is the first to demonstrate the utility of the Luminex system for the immunodetection of both bacteria and soluble markers directly from blood cultures. Targeting both the bacterial pathogens as well as two different disease biomarkers for each infection, we demonstrated the benefit of the multiplexed developed assays for enhanced, reliable detection. The presented arrays could easily be expanded to include antibodies for the detection of other pathogens of interest in hospitals or labs, demonstrating the applicability of this technology for the accurate detection and confirmation of a wide range of potential select agents.

Detection of retromer assembly in Plasmodium falciparum by immunosensing coupled to Surface Plasmon Resonance.

Retromer complex plays a crucial role in intracellular protein trafficking and is conserved throughout the eukaryotes including malaria parasite, Plasmodium falciparum, where it is partially conserved. The assembly of retromer complex in RBC stages of malarial parasite is extremely difficult to explore because of its complicated physiology, small size, and intra-erythrocytic location. Nonetheless, understanding of retromer assembly may pave new ways for the development of novel antimalarials targeting parasite-specific protein trafficking pathways. Here, we investigated the assembly of retromer complex in P. falciparum, by an immunosensing method through highly sensitive Surface Plasmon Resonance (SPR) technique. After taking leads from the bioinformatics search and literature, different interacting proteins were identified and specific antibodies were raised against them. The sensor chip was prepared by covalently linking antibody specific to one component and the whole cell lysate was passed through it in order to trap the interacting complex. Antibodies raised against other interacting components were used to detect them in the trapped complex on the SPR chip. We were able to detect three different components in the retromer complex trapped by the immobilized antibody specific against a different component on a sensor chip. The assay was reproduced and validated in a different two-component CD74-MIF system in mammalian cells. We, thus, illustrate the assembly of retromer complex in P. falciparum through a bio-sensing approach that combines SPR with immunosensing requiring a very small amount of sample from the native source.

Detection and purification of backbone-cyclized proteins using a bacterially expressed anti-myc-tag single chain antibody.

A myc-tag and of which recognition by an antibody 9E10 has long been used for the detection and purification of recombinant proteins. We have previously expanded the application of the tag to the specific detection and purification of backbone-cyclized proteins. Here we sought a more practical way to using the 9E10 antibody by expressing its single chain antibody (scAb) form in Escherichia coli. The combined use of a strong T7 promoter and auto-induction strategy rather than early to mid-log induction of a Lac promoter resulted in the soluble over-expression of 9E10 scAb. However, the co-expression of a chaperone, Skp, was absolutely necessary for the activity even when the protein was expressed in a soluble manner. We could purify about 4 mg of 9E10 scAb from 1 l of culture, and the resulting scAb could be used to detect and purify the backbone-cyclized protein as the parental full-length 9E10. Moreover, the immunoaffinity resin prepared using 9E10 scAb could be regenerated several times after the elution of bound proteins using an acid, which added more value to the ready preparation of the active antibody in bacteria.

A new kind of highly sensitive competitive lateral flow immunoassay displaying direct analyte-signal dependence. Application to the determination of the mycotoxin deoxynivalenol.

A new kind of competitive immunochromatographic assay is presented. It is based on the use of a test strip loaded with (a) labeled specific antibodies, (b) a hapten-protein conjugate at the control zone, and (c) antibodies interacting with the specific antibodies in the analytical zone. In the case where a sample does not contain the target antigen (hapten), all labeled antibodies remain in the control zone because of the selected ratio of reactants. The analytical zone remains colorless because the labeled antibodies do not reach it. If an antigen is present in the sample, it interferes with the binding of the specific antibodies in the control zone and knocks them out. Some of these antibodies pass the control zone to form a colored line in the analytical zone. The intensity of the color is directly proportional to the amount of the target antigen in the sample. The assay has an attractive feature in that an appearance in coloration is more easily detected visually than a decoloration. Moreover, the onset of coloration is detectable at a lower concentration than a decoloration. The new detection scheme was applied to the determination of the mycotoxin deoxynivalenol. The visual limit of detection is 2 ng·mL-1 in corn extracts (35 ng per gram of sample). With the same reagents, this is lower by a factor of 60 than the established test strip. The assay takes only 15 min. This new kind of assay has wide potential applications for numerous low molecular weight analytes. Graphical abstract Competitive immunochromatography with direct analyte-signal dependence is proposed. It provides a 60-fold decrease of the detection limit for mycotoxin deoxynivalenol. The analyte-antibody-label complexes move along the immobilized antigen (control zone) and bind with anti-species antibodies (test zone).

Spotlight on Biomimetic Systems Based on Lyotropic Liquid Crystal.

The behavior of lyotropic biomimetic systems in drug delivery was reviewed. These behaviors are influenced by drug properties, the initial water content, type of lyotropic liquid crystals (LLC), swell ability, drug loading rate, the presence of ions with higher or less kosmotropic or chaotropic force, and the electrostatic interaction between the drug and the lipid bilayers. The in vivo interaction between LCC-drugs, and the impact on the bioavailability of drugs, was reviewed. The LLC with a different architecture can be formed by the self-assembly of lipids in aqueous medium, and can be tuned by the structures and physical properties of the emulsion. These LLC lamellar phase, cubic phase, and hexagonal phase, possess fascinating viscoelastic properties, which make them useful as a dispersion technology, and a highly ordered, thermodynamically stable internal nanostructure, thereby offering the potential as a sustained drug release matrix for drug delivery. In addition, the biodegradable and biocompatible nature of lipids demonstrates a minimum toxicity and thus, they are used for various routes of administration. This review is not intended to provide a comprehensive overview, but focuses on the advantages over non modified conventional materials and LLC biomimetic properties.