Design and Validation of a Three-Dimensional Printer-Based System Enabling Rapid, Low-Cost Construction of the Biosensing Areas of Lateral Flow Devices for Immunoassays and Nucleic Acid Assays.

The COVID-19 pandemic proved the great usefulness of lateral flow tests as self- and rapid tests. The rapid expansion of this field requires the design and validation of novel, affordable, and versatile technologies for the easy fabrication of a variety of lateral flow devices. In the present work, we have developed a new, simple, and cost-effective system for the dispensing of reagents on the membranes of lateral flow devices to be used for research purposes. The 3D printing technology is integrated, for the first time, with simple and inexpensive tools such as a technical pen and disposable pipet tips for the construction of the test and the control areas of the devices. We also used this system for the automated fabrication of spots on the membrane for multiplex analysis. The devices were applied for the detection of proteins/antibodies and single- and double-stranded DNA targets. Also, devices with multiple biosensing areas on the membrane were constructed for the simultaneous detection of different analytes. The proposed system is very simple, automated, and inexpensive and has provided rapid and reproducible construction of lateral flow devices. Compared to a commercially available automated dispenser, the devices showed similar detection capabilities and reproducibility in various real samples. Moreover, contrary to the existing dispensers, the proposed system does not require any gas or costly precision pumps and syringes for the deposition. In conclusion, the developed 3D printer-based system could be an extremely useful alternative for research laboratories for the construction of lateral flow devices of various assay configurations.

Interrogation of endothelial and mural cells in brain metastasis reveals key immune-regulatory mechanisms.

Brain metastasis (BrM) is a common malignancy, predominantly originating from lung, melanoma, and breast cancers. The vasculature is a key component of the BrM tumor microenvironment with critical roles in regulating metastatic seeding and progression. However, the heterogeneity of the major BrM vascular components, namely endothelial and mural cells, is still poorly understood. We perform single-cell and bulk RNA-sequencing of sorted vascular cell types and detect multiple subtypes enriched specifically in BrM compared to non-tumor brain, including previously unrecognized immune regulatory subtypes. We integrate the human data with mouse models, creating a platform to interrogate vascular targets for the treatment of BrM. We find that the CD276 immune checkpoint molecule is significantly upregulated in the BrM vasculature, and anti-CD276 blocking antibodies prolonged survival in preclinical trials. This study provides important insights into the complex interactions between the vasculature, immune cells, and cancer cells, with translational relevance for designing therapeutic interventions.

Reliability of automated brain volumetric analysis: A test by comparing NeuroQuant and volBrain software.

BACKGROUND AND PURPOSE: Brain volume analysis from magnetic resonance imaging (MRI) is gaining an important role in neurological diagnosis. This study compares the volumes of brain segments measured by two automated brain analysis software, NeuroQuant (NQ), and volBrain (VB) in order to test their reliability in brain volumetry. METHODS: Using NQ and VB software, the same brain segment volumes were calculated and compared, taken from 56 patients scanned under the same MRI sequence. These segments were intracranial cavity, putamen, thalamus, amygdala, whole brain, cerebellum, white matter, and hippocampus. The paired t-test method has been used to determine if there was a significant difference in these measurements. The interclass correlation (ICC) is used to test inter-method reliability between the two software. Finally, regression analysis was used to examine the possibility of linear correlation. RESULTS: In all brain segments tested but hippocampus, significant differences were found. ICC presents satisfactory to excellent reliability in all brain segments except thalamus and amygdala for which reliability has been proven to be poor. In most cases, linear correlation was found. CONCLUSIONS: The significant differences found in the majority of the tested brain segments are raising questions about the reliability of automated brain analysis as a quantitative tool. Strong linear correlation of the volumetric measurements and good reliability indicates that, each software provides good qualitative information of brain structures size.

A universal lateral flow assay for microRNA visual detection in urine samples.

MicroRNAs (miRNAs) have been emerged as novel and significant biomarkers in liquid biopsy that can be found in different body fluids. Several techniques have been developed and applied for miRNAs analysis, including nucleic acid-based amplification methods, next generation sequencing, DNA microarrays and new genome-editing methods. These methods, however, are time-consuming and require expensive instruments and specially trained personnel. Biosensors, on the other hand, are alternative and valuable analytical/diagnostic tools due to their simplicity, cost-effectiveness, rapid analysis and ease of use. Several biosensors, especially nanotechnology-based ones, have been developed for miRNA analysis that are based either on target amplification or signal amplification and target re-cycling for sensitive detection. At this point of view, we have introduced a new and universal lateral flow assay in combination with reverse transcription - polymerase chain reaction (RT-PCR) and gold nanoparticles as reporters for the detection of miR-21 and miR-let-7a in human urine. It is the first time that such a biosensor has been applied to the detection of microRNAs in urine. As low as 102-103 copies of miR-21 and 102--104 copies of miR-let-7a added in urine were detectable by the proposed lateral flow assay with great specificity and repeatability (%CVs <4.5%).

Optimization of Silver Nanoparticle Synthesis by Banana Peel Extract Using Statistical Experimental Design, and Testing of their Antibacterial and Antioxidant Properties.

BACKGROUND: In this study, silver nanoparticles (AgNPs) were synthesized using Banana Peel Extract (BPE), and characterized using UV- Vis absorbance spectroscopy, X-Ray Powder Diffraction (XRD), Atomic Force Microscopy (AFM), and Fourier Transform Infrared Spectroscopy (FTIR). UV-Vis absorbance spectroscopy showed the characteristic plasmon resonance of AgNPs at 433 nm. The synthesized AgNPs were tested for their antibacterial and antioxidant properties. METHODS: Nanoparticle size (between 5 and 9 nm) was measured using AFM, whereas their crystallinity was shown by XRD. FTIR identified the ligands that surround the nanoparticle surface. The synthesis conditions were optimised using Central Composite Design (CCD) under Response Surface Methodology (RSM). Silver nitrate (AgNO3) and BPE concentrations (0.25-2.25 mM, 0.2-1.96 % v/v respectively), incubation period (24-120 h) and pH level (2.3-10.1) were chosen as the four independent factors. The fitting parameters (i.e. the wavelength at peak maximum, the peak area, and the peak width) of a Voigt function of the UV- Vis spectra were chosen as the responses. The antibacterial properties of the AgNPs were tested against Escherichia coli and Staphylococcus aureus using the tube dilution test. The synthesized nanoparticles were tested for total phenolic composition (TPC) using the Folin - Ciocalteau method, whereas their radical scavenging activity using the 1,1-diphenyl-2- picrylhydrazyl (DPPH) free radical assay. RESULTS: An optimum combination of all independent factors was identified (BPE concentration 1.7 % v/v, AgNO3 concentration 1.75 mM, incubation period 48 h, pH level 4.3), giving minimum peak wavelength and peak width. The nanoparticles inhibited the growth of E. coli, whereas S. aureus growth was not affected. However, no superiority of AgNPs compared to AgNO3 used for their fabrication (1.75 mM), with respect to antibacterial action, could be here demonstrated. AgNPs were found to present moderate antioxidant activity (44.71± 3.01%), as measured using DPPH assay, while the BPE (used for their fabrication) presented alone (100%) an antioxidant action equal to 86±1%, something expected due to its higher total phenolic content (TPC) compared to that of nanoparticles. CONCLUSION: Altogether, the results of this study highlight the potential of an eco-friendly method to synthesize nanoparticles and its promising optimization through statistical experimental design. Future research on the potential influence of other synthesis parameters on nanoparticles yield and properties could further promote their useful biological activities towards their successful application in the food industry and other settings.