Celiac disease detection using a transglutaminase electrochemical immunosensor fabricated on nanohybrid screen-printed carbon electrodes

Celiac disease is a gluten-induced autoimmune enteropathy characterized by the presence of tissue tranglutaminase (tTG) autoantibodies. A disposable electrochemical immunosensor (EI) for the detection of IgA and IgG type anti-tTG autoantibodies in real patient's samples is presented. Screen-printed carbon electrodes (SPCE) nanostructurized with carbon nanotubes and gold nanoparticles were used as the transducer surface. This transducer exhibits the excellent characteristics of carbon-metal nanoparticle hybrid conjugation and led to the amplification of the immunological interaction. The immunosensing strategy consisted of the immobilization of tTG on the nanostructured electrode surface followed by the electrochemical detection of the autoantibodies present in the samples using an alkaline phosphatase (AP) labelled anti-human IgA or IgG antibody. The analytical signal was based on the anodic redissolution of enzymatically generated silver by cyclic voltammetry. The results obtained were corroborated with a commercial ELISA kit indicating that the electrochemical immunosensor is a trustful analytical screening tool.     

Trace detection of picloram using an electrochemical immunosensor based on three-dimensional gold nanoclusters

Picloram, a herbicide widely used for broadleaf weed control, is persistent and mobile in soil and water with adverse health and environmental effects. It is important to develop a sensitive method for accurate detection of trace picloram in the environment. In this article, a type of ordered three-dimensional (3D) gold (Au) nanoclusters obtained by two-step electrodeposition using the spatial obstruction/direction of the polycarbonate membrane is reported. Bovine serum albumin (BSA)-picloram was immobilized on the 3D Au nanoclusters by self-assembly, and then competitive immunoreaction with picloram antibody and target picloram was executed. The horseradish peroxidase (HRP)-labeled secondary antibody was applied for enzyme-amplified amperometric measurement. The electrodeposited Au nanoclusters built direct electrical contact and immobilization interface with protein molecules without postmodification and positioning. Under the optimal conditions, the linear range for picloram determination was 0.001-10 μg/ml with a correlation coefficient of 0.996. The detection and quantification limits were 5.0 × 10⁻⁴ and 0.0021 μg/ml, respectively. Picloram concentrations in peach and excess sludge supernatant extracts were tested by the proposed immunosensor, which exhibited good precision, sensitivity, selectivity, and storage stability.     

3D cellular spheroids as tools for understanding carboxylated quantum dot behavior in tumors

Background

Monolayer cell cultures have been considered the most suitable technique for in vivo cellular experiments. However, a lot of cellular functions and responses that are present in natural tissues are lost in two-dimensional cell cultures. In this context, nanoparticle accumulation data presented in literature are often not accurate enough to predict behavior of nanoparticles in vivo. Cellular spheroids show a higher degree of morphological and functional similarity to the tissues.

Detection of 2,4,6-trinitrotoluene in seawater using a reversed-displacement immunosensor

Reported in this study are the experimental design and results of an immunosensor for the detection of the explosive, 2,4,6-trinitrotoluene (TNT) in seawater using a reversed-displacement format. This reversed-displacement immunosensor methodology has successfully measured TNT in seawater by direct injection, eliminating the need for preconcentration or pretreatment of samples. A microcolumn containing an Affi-Gel resin derivatized with a 2,4,6-trinitrobenzene (TNB) moiety and a fluorophore-labeled anti-TNT antibody composed the immunoassay reactive chamber. Fluorophore-labeled anti-TNT antibody was incubated with the modified Affi-Gel resin until binding equilibrium was reached. Under a constant flow, samples containing TNT were introduced into the flow stream displacing the fluorophore-labeled TNT antibody. Limits of detection were 2.5ng/mL or part-per-billion (ppb) for TNT in saline buffer and 25ppb in seawater with an analysis time of 10 min. Two anti-TNT antibodies with differing binding affinities were compared in the reversed-displacement assay format, and a correlation between affinity and detection limits was observed. Furthermore, we have demonstrated that the reversed-displacement format can be used to screen seawater samples containing TNT, remains effective after dozens of cycles, and provides significant fluorescence response before regeneration is required.     

Modelling glioma invasion using 3D bioprinting and scaffold-free 3D culture

Glioma is a highly aggressive form of brain cancer, with some subtypes having 5-year survival rates of less than 5%. Tumour cell invasion into the surrounding parenchyma seems to be the primary driver of these poor outcomes, as most gliomas recur within 2 cm of the original surgically-resected tumour. Many current approaches to the development of anticancer therapy attempt to target genetic weaknesses in a particular cancer, but may not take into account the microenvironment experienced by a tumour and the patient-specific genetic differences in susceptibility to treatment. Here we demonstrate the use of complementary approaches, 3D bioprinting and scaffold-free 3D tissue culture, to examine the invasion of glioma cells into neural-like tissue with 3D confocal microscopy. We found that, while both approaches were successful, the use of 3D tissue culture for organoid development offers the advantage of broad accessibility. As a proof-of-concept of our approach, we developed a system in which we could model the invasion of human glioma cells into mouse neural progenitor cell-derived spheroids. We show that we can follow invasion of human tumour cells using cell-tracking dyes and 3D laser scanning confocal microscopy, both in real time and in fixed samples. We validated these results using conventional cryosectioning. Our scaffold-free 3D approach has broad applicability, as we were easily able to examine invasion using different neural progenitor cell lines, thus mimicking differences that might be observed in patient brain tissue. These results, once applied to iPSC-derived cerebral organoids that incorporate the somatic genetic variability of patients, offer the promise of truly personalized treatments for brain cancer.     

Cryosurgery planning using bubble packing in 3D

As part of an ongoing project to develop automated tools for cryosurgery planning, the current study focuses on the development of a 3D bubble packing method. A proof-of-concept for the new method is demonstrated on five prostate models, reconstructed from ultrasound images. The new method is a modification of an established method in 2D. Ellipsoidal bubbles are packed in the volume of the prostate in the current study; such bubbles can be viewed as a first-order approximation of a frozen region around a single cryoprobe. When all cryoprobes are inserted to the same depth, optimum planning was found to occur at about 60% of the length of the prostate (measured from its apex), which leads to cooling of approximately 75% of the prostate volume below a specific temperature threshold of - 22 degrees C. Bubble packing has the potential to dramatically reduce the run time for automated planning.     

Designing of PLA scaffolds for bone tissue replacement fabricated by ordinary commercial 3D printer

Background

The primary objective of Tissue engineering is a regeneration or replacement of tissues or organs damaged by disease, injury, or congenital anomalies. At present, Tissue engineering repairs damaged tissues and organs with artificial supporting structures called scaffolds. These are used for attachment and subsequent growth of appropriate cells. During the cell growth gradual biodegradation of the scaffold occurs and the final product is a new tissue with the desired shape and properties.In recent years, research workplaces are focused on developing scaffold by bio-fabrication techniques to achieve fast, precise and cheap automatic manufacturing of these structures. Most promising techniques seem to be Rapid prototyping due to its high level of precision and controlling. However, this technique is still to solve various issues before it is easily used for scaffold fabrication.In this article we tested printing of clinically applicable scaffolds with use of commercially available devices and materials. Research presented in this article is in general focused on “scaffolding” on a field of bone tissue replacement.

Results

Commercially available 3D printer and Polylactic acid were used to create originally designed and possibly suitable scaffold structures for bone tissue engineering. We tested printing of scaffolds with different geometrical structures. Based on the osteosarcoma cells proliferation experiment and mechanical testing of designed scaffold samples, it will be stated that it is likely not necessary to keep the recommended porosity of the scaffold for bone tissue replacement at about 90%, and it will also be clarified why this fact eliminates mechanical properties issue. Moreover, it is demonstrated that the size of an individual pore could be double the size of the recommended range between 0.2–0.35 mm without affecting the cell proliferation.

Conclusion

Rapid prototyping technique based on Fused deposition modelling was used for the fabrication of designed scaffold structures. All the experiments were performed in order to show how to possibly solve certain limitations and issues that are currently reported by research workplaces on the field of scaffold bio-fabrication. These results should provide new valuable knowledge for further research.

     

Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of botulinum neurotoxin using high-affinity antibodies

A fluorescence sandwich immunoassay using high-affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum neurotoxin serotype A (BoNT/A) using a nontoxic recombinant fragment of the holotoxin (BoNT/A-H_C-fragment) as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. Detection to 31 pM with a total incubation time of 3 h was demonstrated. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the reactions were carried out in microcentrifuge tubes with an incubation time of 1 h. The beads were subsequently captured and concentrated in a rotating rod “renewable surface” flow cell equipped with a fiber optic system for fluorescence measurements. In PBS buffer, the BoNT/A-H_C-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach.     

Analysis of glycoproteins in cell culture supernatants using a lectin immunosensor technique

A method based on a surface plasmon resonance technique for detection of changes in concentration and glycosylation of proteins in cell culture supernatant is described. The method was used to analyze alpha(1)-acid glycoprotein (AGP) produced by a human hepatoma cell line (HepG2). Cell culture supernatant was injected to a BIACORE 2000 instrument and AGP was captured on the sensor chip by immobilized antibodies. The captured glycoprotein was then analyzed for content of carbohydrate epitopes using three different lectins, Aleuria aurantia lectin (AAL), Sambucus nigra agglutinin (SNA), and Triticum vulgaris agglutinin (wheat germ agglutinin, WGA). The method was used to analyze changes in concentration and glycosylation of AGP produced by HepG2 cells grown with or without three different cytokines, interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and transforming growth factor beta-1 (TGF beta(1)). Using the described method it was shown that when HepG2 cells were grown in the presence of IL-6 both AGP concentration and fucosylation increased. When HepG2 cells instead were grown in the presence of TGF beta(1) AGP fucosylation increased whereas AGP concentration decreased.     

Improving wildlife tracking using 3D information

The monitoring of wildlife populations is of growing importance due to the worldwide endangerment of many species, global climate change, and land cover change. Wildlife monitoring by camera traps is an established and non-invasive standard approach to quantify species diversity, estimate occupancy and relative abundance and track animal behaviour in 24/7 documentation. We propose a novel wildlife-specific 3D multi-object tracking workflow using inexpensive stereo camera traps. By embedding carefully efficient 2D methods into the overall 3D workflow, we avoid, on the one hand, costly processing of complex 3D data structures, i.e., 3D point clouds but on the other hand outperform significantly typical 2D tracking approaches with our overall 3D workflow in terms of international established multi-object tracking metrics, i.e., with respect to the reliability and accuracy of the tracking results. The code is available at https://github.com/m-klasen/3d_wildlife-tracking     

Sensitivity improvement in 2D and 3D HCCH spectroscopy using heteronuclear cross-polarization

Summary A new method, which employs a sequence of heteronuclear-homonuclear-heteronuclear Hartmann-Hahn (HEHOHEHAHA) cross-polarization steps for obtaining through-bond H-C-C-H correlations in larger proteins (M_r > 15 kDa), is presented. The method has significantly higher sensitivity compared to INEPTHOHAHA-INEPT-based techniques. An additional feature of this experiment is that well-phaseable spectra may be obtained with a minimal (4-step) phase cycle and, consequently, experimental time can be utilized towards obtaining high resolution in indirect dimensions. Results from 2D and 3D HEHOHEHAHA experiments on T4-lysozyme are presented.     

Quantitative 3D fluorescence technique for the analysis of en face preparations of arterial walls using quantum dot nanocrystals and two-photon excitation laser scanning microscopy

Traditional imaging with one-photon confocal microscopy and organic fluorophores poses several challenges for the visualization of vascular tissue, including autofluorescence, fluorophore crosstalk, and photobleaching. We studied human coronary arteries (HCAs) and mouse aortas with a modified immunohistochemical (IHC) "en face" method using quantum dot (Qdot) bioconjugates and two-photon excitation laser scanning microscopy (TPELSM). We demonstrated the feasibility of multilabeling intimal structures by exciting multicolored Qdots with only one laser wavelength (750 nm). Detailed cell structures, such as the granular appearance of von Willebrand factor (VWF) and the subcellular distribution of endothelial nitric oxide synthase, were visualized using green dots (525 nm), even when the emission maximum of these Qdots overlapped that of tissue autofluorescence (510-520 nm). In addition, sensitive fluorescence quantification of vascular cell adhesion molecule 1 expression at areas of varying hemodynamics (intercostal branches vs. nonbranching areas) was performed in normal C57Bl/6 mice. Finally, we took advantage of the photostability of Qdots and the inherent three-dimensional (3D) resolution of TPELSM to obtain large z-stack series without photobleaching. This innovative en face method allowed simple multicolor profiling, highly sensitive fluorescence quantitation, and 3D visualization of the vascular endothelium with excellent spatial resolution. This is a promising technique to define the spatial and temporal interactions of endothelial inflammatory markers and quantify the effects of different interventions on the endothelium.     

Digital stereophotogrammetry based on circular markers and zooming cameras: evaluation of a method for 3D analysis of small motions in orthopaedic research

Background  

Orthopaedic research projects focusing on small displacements in a small measurement volume require a radiation free, three dimensional motion analysis system. A stereophotogrammetrical motion analysis system can track wireless, small, light-weight markers attached to the objects. Thereby the disturbance of the measured objects through the marker tracking can be kept at minimum. The purpose of this study was to develop and evaluate a non-position fixed compact motion analysis system configured for a small measurement volume and able to zoom while tracking small round flat markers in respect to a fiducial marker which was used for the camera pose estimation.     

An electrochemical immunosensor for aflatoxin M1 determination in milk using screen-printed electrodes

The production and assembling of disposable electrochemical AFM1 immunosensors, which can combine the high selectivity of immunoanalysis with the ease of the electrochemical probes, has been carried out. Firstly immunoassay parameters such as amounts of antibody and labelled antigen, buffer and pH, length of time and temperature of each steps (precoating, coating, binding and competition steps) were evaluated and optimised in order to set up a spectrophotometric enzyme-linked immunosorbent assay (ELISA) procedure. This assay exhibited a working range between 30 and 160 ppt in a direct competitive format. Then electrochemical immunosensors were fabricated by immobilising the antibodies directly on the surface of screen-printed electrodes (SPEs), and allowing the competition to occur between free AFM1 and that conjugated with peroxidase (HRP) enzyme. The electrochemical technique chosen was the chronoamperometry, performed at -100 mV. Furthermore, studies of interference and matrix effects have been performed to evaluate the suitability of the developed immunosensors for the analysis of aflatoxin M1 directly in milk. Results have shown that using screen-printed electrodes aflatoxin M1 can be measured with a detection limit of 25 ppt and with a working range between 30 and 160 ppt. A comparison between the spectrophotometric and electrochemical procedure showed that a better detection limit and shorter analysis time could be achieved using electrochemical detection.     

Combination therapy with BMP-2 and BMSCs enhances bone healing efficacy of PCL scaffold fabricated using the 3D plotting system in a large segmental defect model

The three-dimensional (3D) plotting system is a rapidly-developing scaffold fabrication method for bone tissue engineering. It yields a highly porous and inter-connective structure without the use of cytotoxic solvents. However, the therapeutic effects of a scaffold fabricated using the 3D plotting system in a large segmental defect model have not yet been demonstrated. We have tested two hypotheses: whether the bone healing efficacy of scaffold fabricated using the 3D plotting system would be enhanced by bone marrow-derived mesenchymal stem cell (BMSC) transplantation; and whether the combination of bone morphogenetic protein-2 (BMP-2) administration and BMSC transplantation onto the scaffold would act synergistically to enhance bone regeneration in a large segmental defect model. The use of the combined therapy did increase bone regeneration further as compared to that with monotherapy in large segmental bone defects.     

Fast similarity search for protein 3D structures using topological pattern matching based on spatial relations

Similarity search for protein 3D structures become complex and computationally expensive due to the fact that the size of protein structure databases continues to grow tremendously. Recently, fast structural similarity search systems have been required to put them into practical use in protein structure classification whilst existing comparison systems do not provide comparison results on time. Our approach uses multi-step processing that composes of a preprocessing step to represent geometry of protein structures with spatial objects, a filter step to generate a small candidate set using approximate topological string matching, and a refinement step to compute a structural alignment. This paper describes the preprocessing and filtering for fast similarity search using the discovery of topological patterns of secondary structure elements based on spatial relations. Our system is fully implemented by using Oracle 8i spatial. We have previously shown that our approach has the advantage of speed of performance compared with other approach such as DALI. This work shows that the discovery of topological relations of secondary structure elements in protein structures by using spatial relations of spatial databases is practical for fast structural similarity search for proteins.