A point-of-care biosensor for rapid detection and differentiation of COVID-19 virus (SARS-CoV-2) and influenza virus using subwavelength grating micro-ring resonator.

In the context of continued spread of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 and the emergence of new variants, the demand for rapid, accurate, and frequent detection is increasing. Moreover, the new predominant strain, Omicron variant, manifests more similar clinical features to those of other common respiratory infections. The concurrent detection of multiple potential pathogens helps distinguish SARS-CoV-2 infection from other diseases with overlapping symptoms, which is significant for providing tailored treatment to patients and containing the outbreak. Here, we report a lab-on-a-chip biosensing platform for SARS-CoV-2 detection based on the subwavelength grating micro-ring resonator. The sensing surface is functionalized by specific antibody against SARS-CoV-2 spike protein, which could produce redshifts of resonant peaks by antigen-antibody combination, thus achieving quantitative detection. Additionally, the sensor chip is integrated with a microfluidic chip featuring an anti-backflow Y-shaped structure that enables the concurrent detection of two analytes. In this study, we realized the detection and differentiation of COVID-19 and influenza A H1N1. Experimental results indicate that the limit of detection of our device reaches 100 fg/ml (1.31 fM) within 15 min detecting time, and cross-reactivity tests manifest the specificity of the optical diagnostic assay. Furthermore, the integrated packaging and streamlined workflow facilitate its use for clinical applications. Thus, the biosensing platform presents a promising approach for attaining highly sensitive, selective, multiplexed, and quantitative point-of-care diagnosis and distinction between COVID-19 and influenza.

Strategies for the Covalent Anchoring of a BMP-2-Mimetic Peptide to PEEK Surface for Bone Tissue Engineering.

Researchers in the field of tissue engineering are always searching for new scaffolds for bone repair. Polyetheretherketone (PEEK) is a chemically inert polymer that is insoluble in conventional solvents. PEEK's great potential in tissue engineering applications arises from its ability to not induce adverse reactions when in contact with biological tissues and its mechanical properties, which are similar to those of human bone. These exceptional features are limited by the bio-inertness of PEEK, which causes poor osteogenesis on the implant surface. Here, we demonstrated that the covalent grafting of the sequence (48-69) mapped on the BMP-2 growth factor (GBMP1α) significantly enhances the mineralization and gene expression of human osteoblasts. Different chemical methods were employed for covalently grafting the peptide onto 3D-printed PEEK disks: (a) the reaction between PEEK carbonyls and amino-oxy groups inserted in the peptides' N-terminal sites (oxime chemistry) and (b) the photoactivation of azido groups present in the peptides' N-terminal sites, which produces nitrene radicals able to react with PEEK surface. The peptide-induced PEEK surface modification was assessed using X-ray photoelectron measurements, while the superficial properties of the functionalized material were analyzed by means of atomic force microscopy and force spectroscopy. Live and dead assays and SEM measurements showed greater cell cover on functionalized samples than the control, without any cytotoxicity induction. Moreover, functionalization improved the rate of cell proliferation and the amount of calcium deposits, as demonstrated by the AlamarBlue™ and alizarin red results, respectively. The effects of GBMP1α on h-osteoblast gene expression were assayed using quantitative real-time polymerase chain reaction.

Cancer Cell Invasion of Mammary Organoids with Basal-In Phenotype.

This paper describes mammary organoids with a basal-in phenotype where the basement membrane is located on the interior surface of the organoid. A key materials consideration to induce this basal-in phenotype is the use of a minimal gel scaffold that the epithelial cells self-assemble around and encapsulate. When MDA-MB-231 breast cancer cells are co-cultured with epithelial cells from day 0 under these conditions, cells self-organize into patterns with distinct cancer cell populations both inside and at the periphery of the epithelial organoid. In another type of experiment, the robust formation of the basement membrane on the epithelial organoid interior enables convenient studies of MDA-MB-231 invasion in a tumor progression-relevant direction relative to epithelial cell-basement membrane positioning. That is, the study of cancer invasion through the epithelium first, followed by the basement membrane to the basal side, is realized in an experimentally convenient manner where the cancer cells are simply seeded on the outside of preformed organoids, and their invasion into the organoid is monitored. Interestingly, invasion is more prominent when tumor cells are added to day 7 organoids with less developed basement membranes compared to day 16 organoids with more defined ones.

A Microfluidic Single-Cell Cloning (SCC) Device for the Generation of Monoclonal Cells.

Single-cell cloning (SCC) is a critical step in generating monoclonal cell lines, which are widely used as in vitro models and for producing proteins with high reproducibility for research and the production of therapeutic drugs. In monoclonal cell line generation, the development time can be shortened by validating the monoclonality of the cloned cells. However, the validation process currently requires specialized equipment that is not readily available in general biology laboratories. Here, we report a disposable SCC device, in which single cells can be isolated, validated, and expanded to form monoclonal cell colonies using conventional micropipettes and microscopes. The monoclonal cells can be selectively transferred from the SCC chip to conventional culture plates, using a tissue puncher. Using the device, we demonstrated that monoclonal colonies of actin-GFP (green fluorescent protein) plasmid-transfected A549 cells could be formed in the device within nine days and subsequently transferred to wells in plates for further expansion. This approach offers a cost-effective alternative to the use of specialized equipment for monoclonal cell generation.

Multilayer architecture microfluidic network array for combinatorial drug testing on 3D-cultured cells.

In vitro testing of drug compounds on cell models during the drug development process represents an indispensable step in the initial screening process. Although drug testing on three-dimensional (3D) cultured cells may provide a more accurate prediction of drug efficacy, it is relatively costly and time-consuming to perform compared with conventional 2D cultures due to the thick z-axis of the 3D models. In this study, we have presented a microfluidic platform with integrated pneumatic valves for producing a thin-gel 3D cell culture-based combinatorial drug screening array (3D-µCDS array). The multilayer architecture and microfluidic layout has a smaller device footprint than a single-layer microfluidic channel arrangement, making it well suited to scaling up for high-throughput combinatorial drug screening on 3D cell model. We performed 8 × 8 combination drug screening experiments with the device using two anti-cancer drugs (doxorubicin and paclitaxel) on MDA-MB-231 and MCF-7 breast cancer cell lines for demonstration. Our results indicate that our 3D-µCDS array device allows the successful screening of multiple drug combinations while reducing the operation time and the number of sample/reagents required, making it an ideal tool for general combinatorial drug screening, as well as for applications using valuable tissues and clinical samples.

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Enzyme-Free Dissociation of Neurospheres by a Microfluidic Chip-Based Method.

Neurosphere assay is a common and robust method for identification of neural stem/progenitor cells, but obtaining large numbers of live single cells from dissociated neurospheres is difficult using nonenzymatic methods. Here, we present an enzyme-free method for high-efficiency neurosphere dissociation into single cells using microfluidic device technology. This method allows single cell dissociation of DC115 and KT98 cells with high cell viabilities (80-85 %), single-cell yield (91-95 %), and recovery (75-93 %).

A microfluidic dual-well device for high-throughput single-cell capture and culture.

In vitro culture of single cells facilitates biological studies by deconvoluting complications from cell population heterogeneity. However, there is still a lack of simple yet high-throughput methods to perform single cell culture experiments. In this paper, we report the development and application of a microfluidic device with a dual-well (DW) design concept for high-yield single-cell loading (~77%) in large microwells (285 and 485 µm in diameter) which allowed for cell spreading, proliferation and differentiation. The increased single-cell loading yield is achieved by using sets of small microwells termed "capture-wells" and big microwells termed "culture-wells" according to their utilities for single-cell capture and culture, respectively. This novel device architecture allows the size of the "culture" microwells to be flexibly adjusted without affecting the single-cell loading efficiency making it useful for cell culture applications as demonstrated by our experiments of KT98 mouse neural stem cell differentiation, A549 and MDA-MB-435 cancer cell proliferation, and single-cell colony formation assay with A549 cells in this paper.

Single-cell enzyme-free dissociation of neurospheres using a microfluidic chip.

Obtaining single dissociated cells from neurospheres is difficult using nonenzymatic methods. In this paper we report the development of a microfluidic-chip-based approach that utilizes flow and microstructures to dissociate neurospheres. We show that this microfluidic-chip-based neurosphere-dissociation method can generate high yields of single cells from dissociated neurospheres of mouse KT98 and DC115 cell models (passage number, 3-8; diameter range, 40-250 µm): 90% and 95%, respectively. The microfluidic-chip-dissociated cells had high viabilities (80-85%) and the ability to regrow into neurospheres, demonstrating the applicability of this device to neurosphere assay applications. In addition, the dissociated cells retained their normal differentiation potentials, as shown by their capabilities to differentiate into three neural lineages (neurons, astroglia, and oligodendrocytes) when cultured in differentiation culture conditions. Since this microfluidic-chip-based method does not require the use of enzymatic reagents, the risk of contamination from exogenous substances could be reduced, making it an attractive tool for a wide range of applications where neurosphere dissociation is needed.

A comparison between adipose tissue and dental pulp as sources of MSCs for tooth regeneration.

In this study, several in vivo and in vitro comparisons were performed to test the possibility of using adipose-derived stem cells (ADSCs), a more convenient cell source than dental pulp stem cells (DPSCs), in tooth regeneration. Using an efficient, non-engineering implantation method, we first demonstrated that both implants of ADSCs and DPSCs were able to grow self-assembled new teeth in adult rabbit extraction sockets with high success rate. The stem cells were necessary because the implants grew no tooth without them. A stepwise comparison showed that the regenerated teeth from these two types of adult stem cells were living with nerves and vascular system and remarkably similar to a normal tooth in many details. Further strictly controlled, side-by-side comparisons between the two types of stem cells also showed that the expression patterns of gene markers and the broad differentiation potentials induced by specific methods in vitro were very similar. Although a few differences were found, they did not affect the tested tooth regeneration in vivo or differentiation in vitro. Furthermore, rabbit ADSCs had a higher growth rate and a better senescence resistance in culture. All these findings suggest that ADSCs, one of the richest adult stem cells in mammals, are very similar and useful as DPSCs for regenerative dentistry.

Cloning and expression of hTRF1 in Escherichia coli and preparation of polyclonal antibody / 生物工程学报

Human telomeric repeat binding factor 1(TRF1) contains one Myb-type DNA-binding repeat and an amino-terminal acidic domain. It can bind to the duplex array of TTAGGG repeats at chromosome ends and is shown to be important in preserving genomic stability, maintaining cell proliferative capacity, and blocking the activation of DNA-damage cell cycle checkpoints. Interestingly, the double strand DNA breaks sensor ATM interacts with and phosphorylates Pin2/TRF1 and inhibits its function after DNA damage. Are there some proteins else that can interact with TRF1 and influence its function? In order to analysis the interaction between TRF1 and other proteins, we must prepare the antiserum that can recognize the endogenous TRF1 of cell lysates. TRF1 cDNA was amplified using cDNA Library of HeLa cell by PCR and cloned into pUCm-T vector. Sequence analysis reveals identity to the GenBank report. The TRF1 cDNA was subcloned into expression vector pET-28c(+) and expressed in E. coli as a fusion protein of 65 kD. The recombinant TRF1 can express in the supernatant (about 12.3% in total protein) on the induction of 0.5 mmol/L IPTG at 37 degrees C for 3 hours. Western-blot analysis showed the recombinant protein can react with TRF1 polyclonal antibody sc-6165 (from Santa Cruz Company). His6-TRF1 was purified by Ni(2+) -NTA resin affinity chromatography made by ourselves and showed to be homogeneity in SDS-PAGE. Rabbits were immunized for four times to prepare polyclonal antibody. The unpurified antiserum can recognize the overexpressed TRF1 with myc-tag and the endogenous Pin2/TRF1 of cell lysate by Western-blot at 1:1000 dilution. At 1:400 dilution, the antiserum can interact with endogenous TRF1 by Immunofluorescence cell staining analysis. The endogenous TRF1 in different cell lines, such as HepG2, 803, MCF7 and HeLa, locates in the nucleus. The soluble expression TRF1 and preparation of its antibody lay the foundation to study it further.