Yongqing Xia

Lysozyme mediated calcium carbonate mineralization.

Lysozyme, a major component of egg white proteins, has been speculated to participate in the calcification of avian eggshells. However, its detailed role during the eggshell formation is not well understood. In this work, the influence of lysozyme on the precipitation of CaCO(3) has been investigated using a combined study of FTIR, XRD, and SEM. The precipitation was produced from (NH(4))(2)CO(3) vapor diffusion into CaCl(2) aqueous solution using a specially built chamber. In the absence of lysozyme, hexagonal platelets of vaterite and their spherical aggregates dominated the precipitates during the first 3-12 h crystallization period studied, with the (001) crystal face well expressed in the hexagonal direction. In contrast, calcite was favored to precipitate in the presence of lysozyme during the same period and the effect was found to be proportional to lysozyme concentration. Furthermore, the (110) face of calcite was expressed in addition to the common (104) face, and the morphological modification was also lysozyme concentration dependent. We attributed these phenomena to the selective adsorption of ammonium ions and lysozyme onto different crystal faces. Our findings have clearly revealed the concentration and face dependent role of lysozyme in CaCO(3) precipitation. This, together with the abundance of lysozyme in the uterine fluid, implies its direct contribution to the hierarchical structures of calcite during the initial stage of eggshell formation.

Rapid microwave-assisted synthesis of ultra-bright fluorescent carbon dots for live cell staining, cell-specific targeting and in vivo imaging

Highly fluorescent carbon dots (CDs) with quantum yields up to 96% were rapidly synthesized within 5 min via microwave irradiation with controllable temperature. Multifunctional bioimaging including live cell staining, cell-specific targeting and in vivo imaging were further demonstrated by using high quality and low cost CDs as contrast agents.

Controllable stabilization of poly(N-isopropylacrylamide)-based microgel films through biomimetic mineralization of calcium carbonate

Two types of thermoresponsive microgels, poly(N-isopropylacrylamide) (PNIPAM) microgels and poly(N-isopropylacrylamide-co-acrylic acid) (PNIPAMAC) microgels were synthesized and used as templates for the mineralization of amorphous calcium carbonate (ACC) by diffusion of CO(2) vapor under ambient conditions. Thermosensitive PNIPAM/CaCO(3) hybrid macroscopic hydrogels and micrometer-sized PNIPAMAC/CaCO(3) hybrid microgels were controllably obtained and different mineralization mechanistic processes were proposed. The impact of the loaded CaCO(3) on the size, morphology, stability, and thermosensitivity of the microgels was also analyzed. PNIPAM/CaCO(3) hybrid macrogels had a slight decrease in thermoresponsive phase transition temperature, while PNIPAMAC/CaCO(3) hybrid microgels showed a clear increase in phase transition temperature. The difference reflected different amount and location of ACC in the gel network, causing different interactions with polymer chains. The PNIPAMAC/CaCO(3) microgels formed stable monolayer films on bare silica wafers and glass coverslips upon drying. The microgel films could facilitate the attachment and growth of 3T3 fibroblast cells and their subsequent detachment upon temperature drop from 37 °C to the ambient condition around 20 °C, thus, offering a convenient procedure for cell harvesting.

Thermoresponsive microgel films for harvesting cells and cell sheets.

This work reports the formation of thermoresponsive poly(N-isopropylacrylamide-co-styrene) (PNIPAAmSt) microgel films and their use for cell growth and detachment via temperature stimuli. Thermoresponsive surface films can be conveniently produced by spin-coating or drop-coating of PNIPAAmSt microgel dispersions onto substrates such as glass coverslips, cell culture plates, and flasks, making this technique widely accessible. The thickness, stability, and reversibility of the PNIPAAmSt films coated on silicon wafers with respect to temperature switching were examined by spectroscopic ellipsometry (SE) and atomic force microscopy (AFM). The results unraveled the direct link between thermoreversibility and changes in film thickness and surface morphology, showing reversible hydration and dehydration. Under different coating conditions, well-packed microgel monolayers could be utilized for effective cell recovery and harvesting. Furthermore, cell adhesion and detachment processes were reversible and there was no sign of loss of cell viability during repeated surface attachment, growth, and detachment, showing a mild interaction between cells and thermoresponsive surface. More importantly, there was little deterioration of the packing of the thermoresponsive films or any major loss of microgel particles during reuse, indicating their robustness. These PNIPAAmSt microgel films thus open up a convenient interfacial platform for cell and cell sheet harvesting while avoiding the damage of enzymatic cleavage.

A two-component active targeting theranostic agent based on graphene quantum dots

Using nanotechnology, therapeutics can be combined with diagnostics for cancer treatment. To do this, a targeting ligand, an imaging contrast agent and an anti-tumour therapeutic agent were the minimum requirements for active targeting nanoassemblies. Here we have developed a novel active targeting theranostic agent, made up of just two components, aptamer AS1411 and graphene quantum dots (GQDs). Each component in our agent plays multiple roles. Confocal microscopy using a 488 nm laser shows that this agent has an excellent capability to label tumour cells selectively. On the therapeutic side, this agent induced a synergistic growth inhibition effect towards cancer cells when irradiated with a near infrared laser of 808 nm. The ultra-small size, good biocompatibility, intrinsic stable fluorescence, and near-infrared response character make GQDs a remarkable constituent to build theranostic agents.

Self-assembled two-dimensional thermoresponsive microgel arrays for cell growth/detachment control

Monodisperse poly(N-isopropylacrylamide-styrene) (PNIPAAmSt) microgels with different St/NIPAAm ratios have been synthesized via a one-step surfactant-free emulsion polymerization process. The resulting microgel dispersions were used to fabricate 2D arrays on the surface of silicon wafers/glass coverslips through dip coating. The thermal responsiveness of the PNIPAAmSt microgel arrays was examined by spectroscopic ellipsometry and the results unraveled that the thermoresponsive behavior of the arrays was highly consistent with the microgels dispersed in the bulk, showing high dependence on the content of styrene. The structure of the films varied from nonclose-packed 2D arrays to close-packed 2D arrays, depending on both properties of the microgels and array fabrication conditions. When the weight ratio of styrene was below 40%, the microgel arrays demonstrated effective control for cell growth and detachment across their volume phase transition temperatures (around 28 °C). The extent of swelling of the microgels was the key factor to determine whether the cells could detach from the film easily. For the rather close-packed 2D arrays prepared by the same kind of PNIPAAmSt microgels, the gaps between microgel particles showed no obvious effect on the rate of cell detachment.

Visible and Near-Infrared Dual-Emission Carbogenic Small Molecular Complex with High RNA Selectivity and Renal Clearance for Nucleolus and Tumor Imaging

Fluorescence imaging requires bioselective, sensitive, nontoxic molecular probes to detect the precise location of lesions for fundamental research and clinical applications. Typical inorganic semiconductor nanomaterials with large sizes (>10 nm) can offer high-quality fluorescence imaging due to their fascinating optical properties but are limited to low selectivity as well as slow clearance pathway. We here report an N- and O-rich carbogenic small molecular complex (SMC, MW < 1000 Da) that exhibits high quantum yield (up to 80%), nucleic acid-binding enhanced excitation-dependent fluorescence (EDF), and a near-infrared (NIR) emission peaked at 850 nm with an ultralarge Stokes shift (∼500 nm). SMCs show strong rRNA affinity, and the resulting EDF enhancement allows multicolor visualization of nucleoli in cells for clear statistics. Furthermore, SMCs can be efficiently accumulated in tumor in vivo after injection into tumor-bearing mice. The NIR emission affords high signal/noise ratio imaging for delineating the true extent of tumor. Importantly, about 80% of injected SMCs can be rapidly excreted from the body in 24 h. No appreciable toxicological responses were observed up to 30 days by hematological, biochemical, and pathological examinations. SMCs have great potential as a promising nucleolus- and tumor-specific agent for medical diagnoses and biomedical research.

Fabrication of Patterned Thermoresponsive Microgel Strips on Cell-Adherent Background and Their Application for Cell Sheet Recovery

Interfaces between materials and cells play a critical role in cell biomedical applications. Here, a simple, robust, and cost-effective method is developed to fabricate patterned thermoresponsive poly(N-isopropylacrylamide-co-styrene) microgel strips on a polyethyleneimine-precoated, non-thermoresponsive cell-adherent glass coverslip. The aim is to investigate whether cell sheets could be harvested from these cell-adherent surfaces patterned with thermoresponsive strips comprised of the microgels. We hypothesize that if the cell-to-cell interaction is strong enough to retain the whole cell sheet from disintegration, the cell segments growing on the thermoresponsive strips may drag the cell segments growing on the cell-adherent gaps to detach, ending with a whole freestanding and transferable cell sheet. Critical value concerning the width of the thermoresponsive strip and its ratio to the non-thermoresponsive gap may exist for cell sheet recovery from this type of surface pattern. To obtain this critical value, a series of strip patterns with various widths of thermoresponsive strip and non-thermoresponsive gap were prepared using negative microcontact printing technology, with COS7 fibroblast cells being used to test the growth and detachment. The results unraveled that COS7 cells preferentially attached and proliferated on the cell-adherent, non-thermoresponsive gaps to form patterned cell layers and that they subsequently proliferated to cover the microgel strips to form a confluent cell layer. Intact COS7 cell sheets could be recovered when the width of the thermoresponsive strip is no smaller than that of the non-thermoresponsive gap. Other cells such as HeLa, NIH3T3, 293E, and L929 could grow similarly; that is, they showed initial preference to the non-thermoresponsive gaps and then migrated to cover the entire patterned surface. However, it was difficult to detach them as cell sheets due to the weak interactions within the cell layers formed. In contrast, when COS7 and HeLa cells were cultured successively, they formed the cocultured cell layer that could be detached together. These freestanding patterned cell sheets could lead to the development of more elaborate tumor models for drug targeting and interrogation.

Patterned Thermoresponsive Microgel Surfaces to Control Cell Detachment.

The aim of this work is to examine how adhered individual cells could detach from the patterned, discontinuous thermoresponsive coating substrate and how different patterns in the form of thermoresponsive squares and gaps would affect cell detachment. Microgels prepared from copolymerization of N-isopropylacrylamide and styrene (pNIPAAmSt) were spin-coated on polyethylenimine (PEI) precoated glass coverslips to form a uniform microgel monolayer; then a surface-moisturized PMDS stamp was used to contact the microgel monolayer at room temperature. The thin layer of water on the PDMS stamp surface worked as an ink to penetrate the microgels so that any microgels in direct contact with the wet stamp surface became swollen and could be peeled away, while uncontacted microgels formed patterns. Using this method, various patterns with different thermo-island diameters and gaps could be fabricated. NIH3T3 fibroblast cells were then cultured on these patterns to study their detachment behavior. It was found that cells could detach not only from these discontinuous thermoresponsive coatings, but also from the patterned surfaces with the thermoresponsive area being as low as 20% of the cell spread area.

An Ultra-High Fluorescence Enhancement and High Throughput Assay for Revealing Expression and Internalization of Chemokine Receptor CXCR4.

Revealing chemokine receptor CXCR4 expression, distribution, and internalization levels in different cancers helps to evaluate cancer progression or prognosis and to set personalized treatment strategy. We here describe a sensitive and high-throughput immunoassay for determining CXCR4 expression and distribution in cancer cells. The assay is accessible to a wide range of users in an ordinary lab only by dip-coating poly(styrene-co-N-isopropylacrylamide) spheres on the glass substrate. The self- assembled spheres form three-dimensional photonic colloidal crystals which enhance the fluorescence of CF647 and Alexa Fluor 647 by a factor of up to 1000. CXCR4 in cells is detected by using the sandwich immunoassay, where the primary antibody recognizes CXCR4 and the secondary antibody is labeled with CF647. With the newly established assay, we quantified the total expression of CXCR4, its distribution on the cell membrane and cytoplasm, and revealed their internalization level upon SDF-1α activation in various cancer cells, even for those with extremely low expression level.