In vitro evaluation of bone cell response to novel 3D-printable nanocomposite biomaterials for bone reconstruction.

Critically-sized segmental bone defects represent significant challenges requiring grafts for reconstruction. 3D-printed synthetic bone grafts are viable alternatives to structural allografts if engineered to provide appropriate mechanical performance and osteoblast/osteoclast cell responses. Novel 3D-printable nanocomposites containing acrylated epoxidized soybean oil (AESO) or methacrylated AESO (mAESO), polyethylene glycol diacrylate, and nanohydroxyapatite (nHA) were produced using masked stereolithography. The effects of volume fraction of nHA and methacrylation of AESO on interactions of differentiated MC3T3-E1 osteoblast (dMC3T3-OB) and differentiated RAW264.7 osteoclast cells with 3D-printed nanocomposites were evaluated in vitro and compared with a control biomaterial, hydroxyapatite (HA). Higher nHA content and methacrylation significantly improved the mechanical properties. All nanocomposites supported dMC3T3-OB cells' adhesion and proliferation. Higher amounts of nHA enhanced cell adhesion and proliferation. mAESO in the nanocomposites resulted in greater adhesion, proliferation, and activity at day 7 compared with AESO nanocomposites. Excellent osteoclast-like cells survival, defined actin rings, and large multinucleated cells were only observed on the high nHA fraction (30%) mAESO nanocomposite and the HA control. Thus, mAESO-based nanocomposites containing higher amounts of nHA have better interactions with osteoblast-like and osteoclast-like cells, comparable with HA controls, making them a potential future alternative graft material for bone defect repair.

Extracellular Vesicle (EV) Dot Blotting for Multiplexed EV Protein Detection in Complex Biofluids.

Extracellular vesicles (EVs) are nanoscale vesicles secreted from cells, carrying biomolecular cargos similar to their cells of origin. Measuring the protein content of EVs in biofluids can offer a crucial insight into human health and disease. For example, detecting tumor-derived EVs' protein markers can aid in early diagnosis of cancer, which is life-saving. In order to use these EV proteins for diagnosis, sensitive and multiplexed methods are required. The current methods for EV protein detection typically require large sample consumption due to challenges with sensitivity and often need an EV isolation step for complex biofluid samples such as blood plasma. In this work, we have developed a simple and sensitive method for multiplexed detection of protein markers on EV membrane surfaces, which we call "EV dot blotting", inspired by conventional dot blotting techniques. After optimization of multiple factors such as antibody concentration, blocking reagent, type of 3D membranes, and use of gold nanoparticles for signal enhancement, cancer-cell-derived EVs were spiked in pooled normal human plasma for conducting a multiplexed assay in a microarray format. Without the need of isolating EVs from blood plasma, a limit of detection of 3.1 × 105 EVs/mL or 1863 EVs/sample was achieved for CD9 protein, 4.7 × 104 EVs/mL or 281 EVs/sample for CD24, and 9.0 × 104 EVs/mL or 538 EVs/sample for EpCAM, up to 4 orders of magnitude lower than those of conventional ELISA. This platform offers sensitive, multiplexed, simple, and low-cost EV protein detection directly from complex biofluids with minimal sample consumption, providing a useful tool for multiplexed EV protein quantification for a variety of applications.

Methods for Enhanced Fluorescence Detection of Proteins by using Entrapped Gold Nanoparticles in Membranes.

Measuring protein levels from biofluids can provide important insight into human health and disease during various physiological and pathological conditions. In many situations, sensitive methods are required for protein quantification because at the early stages of many diseases, proteins in biofluids are present at very low concentrations. Here, a new and simple method is presented in the form of Basic and Alternative Protocols for an immunoassay performed on a nitrocellulose membrane, followed by the addition of gold nanoparticles prior to measuring fluorescence with a microscope. The assay protocol was optimized to achieve 3D metal-enhanced fluorescence (MEF) with increased antibody-binding capacity and enhanced fluorescence signals, improving assay sensitivity. Using different concentrations of spiked fluorescently labeled IgGs in pooled normal human plasma, a lower detection limit of 29 ng/ml was achieved. This limit of detection was found to be a thousand-fold lower than the conventional 2D assay and one order of magnitude lower than when the assay was performed on a 3D membrane without MEF. This method provides an easy way to improve immunoassay sensitivity, and it can be simply transferred to other labs. It also can extend to fluorescence detection of other analytes beyond proteins. © 2022 Wiley Periodicals LLC. Basic Protocol: Assay in nitrocellulose membrane with entrapped AuNPs using commercially available AuNPs Alternative Protocol: Assay in nitrocellulose membrane with entrapped AuNPs using lab-made AuNPs.

Entrapping gold nanoparticles in membranes for simple-to-use enhanced fluorescence detection of proteins.

Quantifying proteins under different physiological and pathological conditions can give important insights into human health and disease, since proteins are the functional components of cells. In order to be able to use protein expression levels to diagnose diseases, sensitive protein quantification techniques are required, because some proteins can be present in low concentrations in biofluids at early stages of a disease. Here, a novel and simple-to-implement signal enhancement method for fluorescence-based protein detection is presented, in which an immunoassay was conducted on a nitrocellulose membrane, and a solution of gold nanoparticles was then pipetted onto the membrane before signal acquisition with a fluorescence microscope. The gold nanoparticles were entrapped and adsorbed into the 3D membrane matrix upon drying due to the hydrophobic interactions with the membrane. Through optimizing the concentration and size of the nanoparticles and comparison of different membranes, we were able to achieve metal enhanced fluorescence (MEF). This new gold nanoparticle-based MEF method, together with the 3D membrane platform that improves antibody and gold nanoparticle binding capacity, provided higher signal intensity and assay sensitivity for fluorescence-based protein quantification. Fluorescently-labeled IgG protein was spiked in human plasma with different concentrations, and the lower limit of detection was determined to be 29 ng/mL, three orders of magnitude lower than that of conventional 2D assays and one order of magnitude lower than that of 3D membrane assays without applying gold nanoparticles. This method offers a simple way of improving the sensitivity of fluorescence-based immunoassays that can be easily adopted in a biological lab with basic lab set up. Furthermore, it can be potentially extended to the fluorescence detection of other analytes beyond proteins.

In-silico selection of cancer blood plasma proteins by integrating genomic and proteomic databases.

Blood protein markers have been studied for the clinical management of cancer. Due to the large number of the proteins existing in blood, it is often necessary to pre-select potential protein markers before experimental studies. However, to date there is a lack of automated method for in-silico selection of cancer blood proteins that integrates the information from both genetic and proteomic studies in a cancer-specific manner. In this work, we synthesized both genomic and proteomic information from several open access databases and established a bioinformatic pipeline for in-silico selection of blood plasma proteins overexpressed in specific type of cancer. We demonstrated the workflow of this pipeline with an example of breast cancer, while the methodology was applicable for other cancer types. With this pipeline we obtained 10 candidate biomarkers for breast cancer. The proposed pipeline provides a useful and convenient tool for in-silico selection of candidate blood protein biomarkers for a variety of cancer research.

Pushing the detection limits: strategies towards highly sensitive optical-based protein detection.

Proteins are one of the main constituents of living cells. Studying the quantities of proteins under physiological and pathological conditions can give valuable insights into health status, since proteins are the functional molecules of life. To be able to detect and quantify low-abundance proteins in biofluids for applications such as early disease diagnostics, sensitive analytical techniques are desired. An example of this application is using proteins as biomarkers for detecting cancer or neurological diseases, which can provide early, lifesaving diagnoses. However, conventional methods for protein detection such as ELISA, mass spectrometry, and western blotting cannot offer enough sensitivity for certain applications. Recent advances in optical-based micro- and nano-biosensors have demonstrated promising results to detect proteins at low quantities down to the single-molecule level, shining lights on their capacities for ultrasensitive disease diagnosis and rare protein detection. However, to date, there is a lack of review articles synthesizing and comparing various optical micro- and nano-sensing methods of enhancing the limits of detections of the antibody-based protein assays. The purpose of this article is to critically review different strategies of improving assay sensitivity using miniaturized biosensors, such as assay miniaturization, improving antibody binding capacity, sample purification, and signal amplification. The pros and cons of different methods are compared, and the future perspectives of this research field are discussed.

Recent advances on protein-based quantification of extracellular vesicles.

Extracellular vesicles (EVs) are secreted by almost all cells into the circulatory system and have the important function of intercellular communication. Ranging in size from 50 to 1000 nm, they are further classified based on origin, size, physical properties and function. EVs have shown the potential for studying various physiological and pathological processes, such as characterizing their parent cells with molecular markers that could further signify diseases. Proteins within EVs are the building blocks for the vesicles to function within a biological system. Isolation and proteomic profiling of EVs can advance the understanding of their biogenesis and functions, which can give further insight of how they can be used in clinical settings. However, the nanoscale size of EVs, which is much smaller than that of cells, comprises a major challenge for EV isolation and the characterization of their protein cargos. With the recent advances of bioanalytical techniques such as lab-on-a-chip devices and innovated flow cytometry, the quantification of EV proteins from a small number of vesicles down to the single vesicle level has been achieved, shining light on the promising applications of these small vesicles for early disease diagnosis and treatment monitoring. In this article, we first briefly review conventional EV protein determination technologies and their limitations, followed by detailed description and analysis of emerging technologies used for EV protein quantification, including optical, non-optical, microfluidic, and single vesicle detection methods. The pros and cons of these technologies are compared and the current challenges are outlined. Future perspectives and potential research directions of the EV protein analysis methods are discussed.