Circulating Tumor Cells Adhesion: Application in Biosensors.

The early and non-invasive diagnosis of tumor diseases has been widely investigated by the scientific community focusing on the development of sensors/biomarkers that act as a way of recognizing the adhesion of circulating tumor cells (CTCs). As a challenge in this area, strategies for CTCs capture and enrichment currently require improvements in the sensors/biomarker's selectivity. This can be achieved by understanding the biological recognition factors for different cancer cell lines and also by understanding the interaction between surface parameters and the affinity between macromolecules and the cell surface. To overcome some of these concerns, electrochemical sensors have been used as precise, fast-response, and low-cost transduction platforms for application in cytosensors. Additionally, distinct materials, geometries, and technologies have been investigated to improve the sensitivity and specificity properties of the support electrode that will transform biochemical events into electrical signals. This review identifies novel approaches regarding the application of different specific biomarkers (CD44, Integrins, and EpCAm) for capturing CTCs. These biomarkers can be applied in electrochemical biosensors as a cytodetection strategy for diagnosis of cancerous diseases.

Automated, High-Throughput Screening of Hybrid Elastin-like Polypeptide/Polysaccharide Multilayer Film Deposition.

The self-assembled layer-by-layer technique has attracted a great deal of attention as a method for engineering bio-functional surfaces under mild chemical conditions. The production of multilayer films, starting from newly designed building blocks, may be laborious, considering the inherent limitations for anticipating how minimal changes in the macromolecular composition may impact both film deposition and performance. This paper presents an automated, high-throughput approach to depositing polyelectrolyte multilayers (PEMs) in multiwell plates, enabling the screening of nearly 100 film formulations in the same process. This high-throughput layer-by-layer (HT-LbL) method runs in an affordable, fully commercial platform using Python-coded routines that can be easily adapted for the materials science lab settings. The HT-LbL system was validated by investigating the deposition of polysaccharide-based films in multiwell plates, probing the absorbance signal of ionically stained polyelectrolyte multilayers (PEMs) prepared in one single batch. The HT-LbL method was also used to investigate the deposition of PEMs with a small library of genetically engineered elastin-like polypeptides (ELPs) with different levels of ionizable and hydrophobic amino acid residues. The deposition of ELP/chitosan films was assessed based on the signal of fluorescently labeled species (chitosan or ELP-mCherry), demonstrating that both electrostatic and hydrophobic residues are essential for film buildup. The growth and surface properties of ELP-mCherry/chitosan films also seemed susceptible to the assembly pH, forming a higher film growth and a rougher and more hydrophobic surface for both polyelectrolytes deposited under a low ionization degree. Overall, this study illustrates the challenge of predicting the growth and properties of multilayer films and how the HT-LbL can accelerate the development of multilayer films that demand high levels of testing and optimization.

Three-Dimensional Printing and Its Potential to Develop Sensors for Cancer with Improved Performance.

Cancer is the second leading cause of death globally and early diagnosis is the best strategy to reduce mortality risk. Biosensors to detect cancer biomarkers are based on various principles of detection, including electrochemical, optical, electrical, and mechanical measurements. Despite the advances in the identification of biomarkers and the conventional 2D manufacturing processes, detection methods for cancers still require improvements in terms of selectivity and sensitivity, especially for point-of-care diagnosis. Three-dimensional printing may offer the features to produce complex geometries in the design of high-precision, low-cost sensors. Three-dimensional printing, also known as additive manufacturing, allows for the production of sensitive, user-friendly, and semi-automated sensors, whose composition, geometry, and functionality can be controlled. This paper reviews the recent use of 3D printing in biosensors for cancer diagnosis, highlighting the main advantages and advances achieved with this technology. Additionally, the challenges in 3D printing technology for the mass production of high-performance biosensors for cancer diagnosis are addressed.

Silk fibroin/nanohydroxyapatite hydrogels for promoted bioactivity and osteoblastic proliferation and differentiation of human bone marrow stromal cells.

Silk fibroin (SF) is a natural, biocompatible, and biodegradable polymer having a great potential for the successful regeneration of damaged bone tissue. In the present work, nanohydroxyapatite (nanoHA) was incorporated into SF polymer to form a bioactive composite hydrogel for applications as bone implants. The degradation and bioactive properties of SF/nanoHA composite hydrogels were evaluated. Additionally, biological investigations of human bone marrow stromal cells (hBMSCs) viability, proliferation and differentiation to the osteoblastic phenotype were conducted. The incorporation of nanoHA in SF polymer matrices improved the bioactivity of the hydrogels. The biological results highlighted that the SF/nanoHA composite hydrogels are suitable for hBMSCs attachment and proliferation, while a test for alkaline phosphatase (ALP) and bone morphogenetic protein 2 (BMP-2) expression suggested osteoblast differentiation. Additionally, a cell staining method for ALP allowed to observe cell infiltration with active production of ALP by the infiltrated cells, paving the way to use the proposed composite hydrogel for bone tissue regeneration.

Nanofilms of hyaluronan/chitosan assembled layer-by-layer: An antibacterial surface for Xylella fastidiosa.

In this work, nanofilms of hyaluronan/chitosan (HA/CHI) assembled layer by layer were synthesized; their application as a potential antimicrobial material was demonstrated for the phytopathogen Xylella fastidiosa, a gram-negative bacterium, here used as a model. For the synthesis, the influence of pH and ionic strength of these natural polymer stem-solutions on final characteristics of the HA/CHI nanofilms was studied in detail. The antibacterial effect was evaluated using widefield fluorescence microscopy. These results were correlated with the chemical properties of the nanofilms, studied by FTIR and Raman spectroscopy, as well as with their morphology and surface properties characterized using SEM and AFM. The present findings can be extended to design and optimize HA/CHI nanofilms with enhanced antimicrobial behavior for other type of phytopathogenic gram-negative bacteria species, such as Xanthomonas citri, Xanthomas campestri and Ralstonia solanacearum.

Liposome-Loaded Cell Backpacks.

Cell backpacks, or micron-scale patches of a few hundred nanometers in thickness fabricated by layer-by-layer (LbL) assembly, are potentially useful vehicles for targeted drug delivery on the cellular level. In this work, echogenic liposomes (ELIPs) containing the anticancer drug doxorubicin (DOX) are embedded into backpacks through electrostatic interactions and LbL assembly. Poly(allylamine hydrochloride)/poly(acrylic acid) (PAH/PAA)n , and poly(diallyldimethylammonium chloride)/poly(styrene sulfonate) (PDAC/SPS)n film systems show the greatest ELIP incorporation of the films studied while maintaining the structural integrity of the vesicles. The use of ELIPs for drug encapsulation into backpacks facilitates up to three times greater DOX loading compared to backpacks without ELIPs. Cytotoxicity studies reveal that monocyte backpack conjugates remain viable even after 72 h, demonstrating promise as drug delivery vehicles. Because artificial vesicles can load many different types of drugs, ELIP containing backpacks offer a unique versatility for broadening the range of possible applications for cell backpacks.

Optimization of amine-rich multilayer thin films for the capture and quantification of prostate-specific antigen.

It is demonstrated that poly(allylamine hydrochloride)/poly(styrenesulfonate) (PAH/SPS) multilayer films can be successfully tailored for the capture and detection of small biomolecules in dilute concentrations. Based on in vitro results, these films could be potentially applied for rapid and high-throughput diagnosis of dilute biomarkers in serum or tissue. PAH presents functional amino groups that can be further reacted with desired chemistries in order to create customizable and specific surfaces for biomolecule capture. A variety of film assembly characteristics were tested (pH, molecular weight of PAH, and ionic strength) to tune the biotinylation and swelling behavior of these films to maximize detection capabilities. The resultant optimized biotinylated PAH/SPS 9.3/9.3 system was utilized in conjunction with quantum dots (Qdots) to capture and detect a dilute biomarker for prostate cancer, prostate-specific antigen (PSA). Compared to previous work, our system presents a good sensitivity for PSA detection within the clinically relevant range of 0.4-100 ng/mL.

Bioactive polyelectrolyte multilayers: hyaluronic acid mediated B lymphocyte adhesion.

A strategy was developed to produce thin, biopolymer-based polyelectrolyte multilayer films, based on hyaluronic acid and chitosan, that are able to effectively bind B lymphocytes. These films explore CD44-hyaluronate interactions and provide a method to make surface-bound B cell arrays without the need for nonselective covalent chemistry. The rational design of these films using solution deposition variables, such as ionic strength and pH, allows one to maximize and fine tune this binding efficiency ex vivo. This work suggests two important conditions for successfully attaching B cells to hyaluronate-containing polyelectrolyte multilayer films: (1) hyaluronic acid is required for the proposed CD44-mediated binding mechanism, and (2) hyaluronic acid deposition conditions that favor loops and tails, such as low pH and with added salt, result in more available CD44 binding ligands and higher cell binding efficiency. Chitosan-terminated films prepared without NaCl in the deposition solutions and hyaluronic acid-terminated films prepared with salt, both under pH 3.0 assembly conditions, presented a similar high lymphocyte binding efficiency. In the former case, however, the binding strength was weaker due to a significant electrostatic contribution to the binding. Bioactive polyelectrolyte multilayers for selective binding of lymphocytes hold great promise in fields ranging from cell-based biosensors to immune system engineering.

Natural and prosthetic heart valve calcification: morphology and chemical composition characterization.

Calcification is the most common cause of damage and subsequent failure of heart valves. Although it is a common phenomenon, little is known about it, and less about the inorganic phase obtained from this type of calcification. This article describes the scanning electron microscopy (SEM)/energy dispersive X-ray spectroscopy and Ca K-edge X-ray absorption near edge structure (XANES) characterization performed in natural and bioprosthetic heart valves calcified in vivo (in comparison to in vitro-calcified valves). SEM micrographs indicated the presence of deposits of similar morphology, and XANES results indicate, at a molecular level, that the calcification mechanism of both types of valves are probably similar, resulting in formation of poorly crystalline hydroxyapatite deposits, with Ca/P ratios that increase with time, depending on the maturation state. These findings may contribute to the search for long-term efficient anticalcification treatments.

Lyophilized bovine pericardium treated with a phenethylamine-diepoxide as an alternative to preventing calcification of cardiovascular bioprosthesis: preliminary calcification results.

This study investigated the calcification process that occurred on chemically treated bovine pericardium substrata through tests with simulated body fluid solutions. The use of bovine pericardium bioprosthetic valves in heart valve surgery has a significant drawback due to the calcification processes. Thus, many routes such as chemical treatments in the substratum or the adoption of systemic therapies are considered in the literature with the intention to inhibit or to decelerate this process. The presented treatment using the two different phenetylamine-diepoxide solutions showed no effects on calcification experiments as showed by the tests. However, the lyophilized bovine pericardium samples, treated with both solutions, did not show any detectable phosphate deposits. The lyophilization of bovine pericardium before chemical treatments with cross-link agents as epoxy compounds may be an alternative to the conventional calcification prevention methods, but further investigations are recommended to check if the same behavior is found in all lyophilized systems.