This work presents potential applications of low-cost fused deposition modeling 3D-printers to fabricate multiuse 3D-printed electrochemical cells for flow or batch measurements as well as the ...3D-printing of electrochemical sensing platforms. Electrochemical cells and sensors were printed with acrylonitrile butadiene styrene (ABS) and conductive graphene-doped polylactic acid (G-PLA) filaments, respectively. The overall printing operation time and estimated cost per cell were 6 h and $ 6.00, respectively, while the sensors were printed within minutes (16 sensor strips of 1 × 2 cm in 10 min at a cost of $ 1.00 each sensor). The cell performance is demonstrated for the amperometric detection of tert-butylhydroquinone, dipyrone, dopamine and diclofenac by flow-injection analysis (FIA) and batch-injection analysis (BIA) using different working electrodes, including the proposed 3D-printed sensor, which presented comparable electroanalytical performance with other carbon-based electrodes (LOD of 0.1 μmol L−1 for dopamine). Raman spectroscopy and scanning electron microscopy of the 3D-printed sensor indicated the presence of graphene nanoribbons within the polymeric matrix. Electrochemical impedance spectroscopy and heterogeneous electron transfer constants (k0) for the redox probe Ru(NH3)6+3 revealed that a glassy-carbon electrode presented faster electron transfer rates than the 3D-printed sensor; however, the latter presented lower LOD values for dopamine and catechol probably due to oxygenated functional groups at the G-PLA surface.
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•Low-cost fused deposition modeling (FDM) 3D-printers to produce cells and electrodes.•Multiuse cells for flow- (FIA) and batch-injection analysis (BIA) as well for batch condition.•Designs and printing conditions accessible for any FDM 3D-printers.•Graphene-doped PLA printed sensors for voltammetric and amperometric detection.•Electroanalytical performance similar to GCE modified with carbon nanomaterials.
This current review article focuses on recent contributions to on-site forensic investigations. Portable and potentially portable methods are presented and critically discussed about (bio)chemical ...trace analysis and studies performed outside the controlled laboratory environment to rapidly help in crime scene inquiries or forensic intelligence purposes. A wide range of approaches including electrochemical sensors, microchip electrophoresis, ambient ionization on portable mass spectrometers, handheld Raman and NIR instruments as well as and point-of-need devices, like paper-based platforms, for in-field analysis of latent evidences, controlled substances, drug screening, hazards, and others to assist in law enforcements and solving crime more efficiently are highlighted. The covered examples have successfully demonstrated the huge potential of portable devices for on-site applications. Future investigations should consider analytical validation to compete equality and even replace current gold standard methods.
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•Electrochemical sensors offer good sensitivity for abuse drugs and explosives.•Paper-based devices have revealed desirable performance for point-of-care testing.•NIR and RAMAN instruments have allowed fast screening at the point-of-need.•Portable MS instruments have exhibited good performance for on-site forensic applications.•Electrophoresis chips have provided excellent ability for STR genotyping.
Summary
Background
Eosinophils, a central factor in asthma pathogenesis, have the ability to secrete exosomes. However, the precise role played by exosomes in the biological processes leading up to ...asthma has not been fully defined.
Objective
We hypothesized that exosomes released by eosinophils contribute to asthma pathogenesis by activating structural lung cells.
Methods
Eosinophils from asthmatic patients and healthy volunteers were purified from peripheral blood, and exosomes were isolated from eosinophils of asthmatic and healthy individuals. All experiments were performed with eosinophil‐derived exosomes from healthy and asthmatic subjects. Epithelial damage was evaluated using primary small airway epithelial cell lines through 2 types of apoptosis assays, that is, flow cytometry and TUNEL assay with confocal microscopy. Additionally, the epithelial repair was analysed by performing wound healing assays with epithelial cells. Functional studies such as proliferation and inhibition‐proliferation assays were carried out in primary bronchial smooth muscle cell lines. Also, gene expression analysis of pro‐inflammatory molecules was evaluated by real‐time PCR on epithelial and muscle cells. Lastly, protein expression of epithelial and muscle cell signalling factors was estimated by Western blot.
Results
Asthmatic eosinophil‐derived exosomes induced an increase in epithelial cell apoptosis at 24 hour and 48 hour, impeding wound closure. In addition, muscle cell proliferation was increased at 72 hours after exosome addition and was linked with higher phosphorylation of ERK1/2. We also found higher expression of several genes when both cell types were cultured in the presence of exosomes from asthmatics: CCR3 and VEGFA in muscle cells, and CCL26, TNF and POSTN in epithelial cells. Healthy eosinophil‐derived exosomes did not exert any effect over these cell types.
Conclusions and Clinical Relevance
Eosinophil‐derived exosomes from asthmatic patients participate actively in the development of the pathological features of asthma via structural lung cells.
Metal–organic frameworks (MOFs), network structures wherein metal ions or clusters link organic ligands into porous materials, are being actively researched as nanoscale drug delivery devices as they ...offer tunable structures with high cargo loading that can easily be further functionalized for targeting and enhanced physiological stability. The excellent biocompatibility of Zr has meant that its MOFs are among the most studied to date, in particular the archetypal Zr terephthalate UiO-66. In contrast, the isoreticular analog linked by fumarate (Zr-fum) has received little attention, despite the endogenous linker being part of the Krebs cycle. Herein, we report a comprehensive study of Zr-fum in the context of drug delivery. Reducing particle size is shown to increase uptake by cancer cells while reducing internalization by macrophages, immune system cells that remove foreign objects from the bloodstream. Zr-fum is compatible with defect loading of the drug dichloroacetate (DCA) as well as surface modification during synthesis, through coordination modulation and postsynthetically. DCA-loaded, PEGylated Zr-fum shows selective in vitro cytotoxicity toward HeLa and MCF-7 cancer cells, likely as a consequence of its enhanced caveolae-mediated endocytosis compared to uncoated precursors, and it is well tolerated by HEK293 kidney cells, J774 macrophages, and human peripheral blood lymphocytes. Compared to UiO-66, Zr-fum is more efficient at transporting the drug mimic calcein into HeLa cells, and DCA-loaded, PEGylated Zr-fum is more effective at reducing HeLa and MCF-7 cell proliferation than the analogous UiO-66 sample. In vitro examination of immune system response shows that Zr-fum samples induce less reactive oxygen species than UiO-66 analogs, possibly as a consequence of the linker being endogenous, and do not activate the C3 and C4 complement cascade pathways, suggesting that Zr-fum can avoid phagocytic activation. The results show that Zr-fum is an attractive alternative to UiO-66 for nanoscale drug delivery, and that a wide range of in vitro experiments is available to greatly inform the design of drug delivery systems prior to early stage animal studies.
•Fused deposition modeling 3D-printed electrode for (bio)sensors applied to real samples.•Enzymatic glucose biosensing on 3D-printed graphene-PLA electrode in plasma.•Oxygenated groups from PLA ...matrix favored enzyme immobilization by crosslinking.•Graphene-PLA 3D-printed electrochemical response improves after surface treatment.•Rapid and precise analysis of urine and saliva by pulsed amperometry using flow system.
Additive manufacturing, also known as 3D-printing, is receiving great interest by chemists due to the easy design of novel materials, fast prototyping and reducing waste, which enables large-scale fabrication of electrochemical devices. Herein we demonstrate the development of (bio)sensors for the analysis of biological fluids using 3D-printing. Fused deposition modelling was used to fabricate (bio)sensing platforms from commercially-available filaments made of polylactic acid containing graphene (G-PLA). An enzymatic glucose biosensor fabricated on the G-PLA surface was developed and applied for glucose sensing in blood plasma using chronoamperometry. Oxygenated groups from the polymeric matrix provides suitable condition to enzyme immobilization by crosslinking with glutaraldehyde. The biosensor presented a limit of detection (LOD) of 15 μmol L−1, inter-day and intra-day precision lower than 5 %, and adequate recovery values (90–105 %) for the analysis of plasma. We also show that the surface treatment of the 3D-printed sensor (mechanical polishing followed solvent immersion) provides improved electrochemical properties for the direct detection of nitrite and uric acid. Differential-pulse voltammetry and multiple-pulse amperometry under flow conditions were evaluated and compared for the determination of both species in saliva and urine. Highlights are presented for the amperometric detection within a linear range from 0.5–250 μmol L−1 for both analytes, LODs of 0.02 and 0.03 μmol L−1 for uric acid and nitrite, respectively, and high precision (RSD < 2.1 %). This report shows the first application of 3D-printed sensors and biosensors for the analysis of real biological samples with analytical features comparable to conventional modified electrodes.
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•Low-cost fused deposition modeling 3D-printed device for sampling and detection of TNT.•Graphene-doped polylacic acid (G-PLA) filament to fabricate the 3D-printed device.•Nanograms ...of TNT sampled from metallic, granite and glove surfaces were quantified.•Mechanical polishing of the 3D-printed surface improved the electrochemical properties.•Metal determination on the device was also shown; promising for gunshot residue analysis.
Fused deposition modelling 3D printing of a flexible, conductive, disposable and biodegradable platform using graphene-doped polylactic acid (G-PLA) was demonstrated as an integrated device for sampling and detection of explosives. As a proof-of-concept, traces of 2,4,6-Trinitrotoluene (TNT) impregnated on different surfaces were abrasively sampled using the 3D-printed device and readily assembled in a portable electrochemical cell for rapid square-wave voltammetry scans in the presence of 0.1 mol L–1 HCl electrolyte. Nanogram amounts of TNT sampled from metallic, granite and glove surfaces were detected and quantified using the Faraday equation applied to the voltammetric response of TNT immobilised on the electrode surface. Identification of TNT was possible due to the unique voltammetric behaviour obtained on the G-PLA sensor and efficient sampling due to the rough surface and flexibility of the device. Lead and copper determination by stripping voltammetry was also demonstrated on the same device, highlighting the possibility of detecting gunshot residues. Moreover, we demonstrated that simple mechanical polishing of the 3D-printed surface improved the electrochemical sensing properties of the sensor by exposing graphene nanoribbons within the PLA matrix. Hence, this 3D-printed integrated platform holds promise as a rapid and low-cost approach for on-site crime scene investigations.
Background
Asthma is a syndrome characterized by airway inflammation and obstruction. Due to its heterogeneity, the difficulties in asthma diagnosis and treatment make the discovery of new biomarkers ...a focus of research. So, we determined the differential miRNA expression of eosinophils between healthy and asthmatic patients and to establish a differentially expressed miRNA profile detectable in sera for use as biomarker.
Methods
MicroRNAs from peripheral eosinophils from healthy and asthmatic subjects were isolated and analyzed by next‐generation sequencing and confirmed by quantitative PCR in 29 asthmatics and 10 healthy individuals. The levels of serum miRNAs were performed by quantitative PCR in 138 asthmatics and 39 healthy subjects. Regression analysis and Random Forest models were performed.
Results
We found a set of miRNAs whose expression differs between eosinophils from asthmatics and healthy subjects. These miRNAs can classify asthmatics into two clusters that differed in the number of eosinophils and periostin concentration in serum. Some of these miRNAs were also confirmed in sera, as miR‐185‐5p which discriminates asthmatics from healthy subjects. Together with other two miRNAs, miR‐185‐5p allowed us to create a logistic regression model to discriminate better both conditions and a Random Forest model that can even sort the asthmatics into intermittent, mild persistent, moderate persistent, and severe persistent asthma.
Conclusion
Our data show that miRNAs profile in eosinophils can be used as asthma diagnosis biomarker in serum and that this profile is able to rank asthma severity.
Eosinophils from asthmatics present a different profile in microRNAs (miRNAs) compared to eosinophils from healthy subjects. As eosinophils obtention from patients is not a standardized method, we analyzed these miRNAs in serum showing that miRNAs profile expression in this biofluid can be used for asthma diagnosis and for severity classification.
Chronic respiratory diseases (CRDs) are an important factor of morbidity and mortality, accounting for approximately 6% of total deaths worldwide. The main CRDs are asthma and chronic obstructive ...pulmonary disease (COPD). These complex diseases have different triggers including allergens, pollutants, tobacco smoke, and other risk factors. It is important to highlight that although CRDs are incurable, various forms of treatment improve shortness of breath and quality of life. The search for tools that can ensure accurate diagnosis and treatment is crucial. MicroRNAs (miRNAs) are small non-coding RNAs and have been described as promising diagnostic and therapeutic biomarkers for CRDs. They are implicated in multiple processes of asthma and COPD, regulating pathways associated with inflammation, thereby showing that miRNAs are critical regulators of the immune response. Indeed, miRNAs have been found to be deregulated in several biofluids (sputum, bronchoalveolar lavage, and serum) and in both structural lung and immune cells of patients in comparison to healthy subjects, showing their potential role as biomarkers. Also, miRNAs play a part in the development or termination of histopathological changes and comorbidities, revealing the complexity of miRNA regulation and opening up new treatment possibilities. Finally, miRNAs have been proposed as prognostic tools in response to both conventional and biologic treatments for asthma or COPD, and miRNA-based treatment has emerged as a potential approach for clinical intervention in these respiratory diseases; however, this field is still in development. The present review applies a systems biology approach to the understanding of miRNA regulatory networks in asthma and COPD, summarizing their roles in pathophysiology, diagnosis, and treatment.
Asthma is a chronic respiratory disease produced by an aberrant immune response that originates with breathing difficulties and cough, through airway remodeling. The above pathophysiological events ...of asthma emerge the regulators of effectors, like epigenetics, which include microRNAs (miRNAs) who perform post‐transcriptional regulation, controlling diverse pathways in respiratory diseases. The objective of the study was to determine how miR‐185‐5p regulates the secretion of periostin by airway structural cells, and smooth muscle cells contraction, both related to airway remodeling in asthma. We used miR‐185‐5p mimic and inhibitors in bronchial smooth muscle cells (BSMCs) and small airway epithelial cells (SAECs) from healthy subjects. Gene expression and protein levels of periostin (POSTN), CDC42, and RHOA were analyzed by RT‐PCR and ELISA/Western blot, respectively. BSMC contractility was analyzed using cell‐embedded collagen gels and measurement of intracellular calcium was performed using Fura‐2. Additionally, miR‐185‐5p and periostin expression were evaluated in sputum from healthy and asthmatics. From these experiments, we observed that miR‐185‐5p modulation regulates periostin mRNA and protein in BSMCs and SAECs. A tendency for diminished miR‐185‐5p expression and higher periostin levels was seen in sputum cells from asthmatics compared to healthy, with an inverse correlation observed between POSTN and miR‐185‐5p. Inhibition of miR‐185‐5p produced higher BSMCs contraction induced by histamine. Calcium mobilization was not modified by miR‐185‐5p, showing that miR‐185‐5p role in BSMC contractility is performed by regulating CDC42 and RhoA pro‐contractile factors instead. In conclusion, miR‐185‐5p is a modulator of periostin secretion by airway structural cells and of smooth muscle contraction, which can be related to asthma pathophysiology, and thus, might be a promising therapeutic target.
In this study, we show that miR‐185‐5p, which is inversely correlated to POSTN expression in sputum from the asthmatic and healthy, is able to modulate periostin secretion by airway structural cells such as epithelial and smooth muscle cells, of crucial importance in airway remodeling, while also being able to regulate the synthesis of RhoA and CDC42 protein levels, which increase smooth muscle contractile capacity. These events are the key mechanisms involved in the development and maintenance of the symptoms of asthma.
The high drug-loading and excellent biocompatibilities of metal–organic frameworks (MOFs) have led to their application as drug-delivery systems (DDSs). Nanoparticle surface chemistry dominates both ...biostability and dispersion of DDSs while governing their interactions with biological systems, cellular and/or tissue targeting, and cellular internalization, leading to a requirement for versatile and reproducible surface functionalization protocols. Herein, we explore not only the effect of introducing different surface functionalities to the biocompatible Zr-MOF UiO-66 but also the efficacy of three surface modification protocols: (i) direct attachment of biomolecules folic acid (FA) and biotin (Biot) introduced as modulators for UiO-66 synthesis, (ii) our previously reported “click-modulation” approach to covalently attach polymers poly(ethylene glycol) (PEG), poly-l-lactide, and poly-N-isopropylacrylamide to the surface of UiO-66 through click chemistry, and (iii) surface ligand exchange to postsynthetically coordinate FA, Biot, and heparin to UiO-66. The innovative use of a small molecule with metabolic anticancer activity, dichloroacetate (DCA), as a modulator during synthesis is described, and it is found to be compatible with all three protocols, yielding surface-coated, DCA-loaded (10–20 w/w %) nano-MOFs (70–170 nm). External surface modification generally enhances the stability and colloidal dispersion of UiO-66. Cellular internalization routes and efficiencies of UiO-66 by HeLa cervical cancer cells can be tuned by surface chemistry, and anticancer cytotoxicity of DCA-loaded MOFs correlates with the endocytosis efficiency and mechanisms. The MOFs with the most promising coatings (FA, PEG, poly-l-lactide, and poly-N-isopropylacrylamide) were extensively tested for selectivity of anticancer cytotoxicity against MCF-7 breast cancer cells and HEK293 healthy kidney cells as well as for cell proliferation and reactive oxygen species production against J774 macrophages and peripheral blood lymphocytes isolated from the blood of human donors. DCA-loaded, FA-modified UiO-66 selectively kills cancer cells without harming healthy ones or provoking immune system response in vitro, suggesting a significant targeting effect and great potential in anticancer drug delivery. The results provide mechanistic insight into the design and functionalization of MOFs for drug delivery and underline the availability of various in vitro techniques to potentially minimize early-stage in vivo animal studies following the three Rs: reduction, refinement, and replacement.
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