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The negative photoresist SU-8 has found widespread use as a material in the fabrication of microelectrical--mechanical systems (MEMS). Although SU-8 has been utilized as a structural material for biological MEMS, a number of SU-8 properties limit its application in these bioanalytical devices. These attributes include its brittleness, nonspecific adsorption of biomolecules, and high fluorescence in the visible wavelengths. In addition, native SU-8 is a poor substrate for cellular adhesion. Photoresists composed of resins with epoxide side groups and photoacids were screened for their ability to serve as a low-fluorescence photoresist with sufficient resolution to generate microstructures with dimensions of 5-10 [micro]m. The fluorescence of structures formed from 1002F photoresist (1002F resin combined with triarylsulfonium hexafluoroantimonate salts) was as much as 10 times less fluorescent than similar SU-8 microstructures. The absorbance of 1002F in the visible wavelengths was also substantially lower than that of SU-8. Microstructures or pallets with an aspect ratio as high as 4:1 could be formed permitting 1002F to be used as a structural material in the fabrication of arrays of pallets for sorting adherent cells. Several different cell types were able to adhere to native 1002F surfaces, and the viability of these cells was excellent. As with SU-8, 1002F has a weak adhesion to glass, a favorable attribute when the pallet arrays are used to sort adherent cells. A threshold, laser pulse energy of 3.5 [micro]J was required to release individual 50 [micro]m, 1002F pallets from an array. Relative to SU-8, 1002F photoresist offers substantial improvements as a substrate in bioanalytical devices and is likely to find widespread use in BioMEMS.

Arrays of micropallets have been used to pattern adherent cells as well to sort mixtures of cells. These artificial surfaces are composed of micrometer-sized, SU-8 structures formed on a hydrophobic glass surface. Successful application of these arrays requires stable Cassie-Baxter wetting by aqueous biological solutions. This paper systematically studies the factors governing the stability of Cassie-Baxter wetting on the arrays, including the surface properties of the array components as well as the physical and chemical properties of the wetting solutions. To establish stable Cassie-Baxter wetting with water, the surface of the array must be coated with a perfluoroalkylsilane of sufficient hydrophobicity, and the roughness of array must be greater than 1.6. Additionally, long-term stability of the Cassie-Baxter wetting depends on the properties of the wetting solutions, including the surface tension (>40 mM/m), salt concentration (> 10 mM), and protein concentration (

The ability to selectively grow out individual cells possessing unique characteristics from within a mixed population is of widespread importance for biomedical investigations. Generation of genetically engineered cell lines, transformation studies, cell-based assays, and stem cell studies are examples where single-cell cloning is of immense value. The vast majority of mammalian cells grow adherent to a surface; therefore, positive selection followed by cloning of cells while the cells remain adherent to their growth surface is an important goal. We recently demonstrated a microfabricated cell array combined with laser-based release of individual array elements for positive selection of single cells. In the current work, a strategy to collect single cells for clonal expansion is described. The system enabled cloning of individual cells with 80-90% efficiency. Single cells were selected and cloned from small populations of fewer than 10 000 cells. Strategies used by cells to migrate from the pallets to form colonies on the surface of the collection device were examined. Implementation of encoded array elements made it possible to follow specific cells throughout the selection, collection, and cloning procedure. Thus, a particular cell can be identified by any number of imaging techniques, isolated, and clonally expanded to generate a homogeneous cell line or a pure sample for genetic or biochemical analysis.

The selection and collection of single cells from within a heterogeneous population is required to produce genetically engineered cell lines, to develop new stem cell lines, and for single-cell studies. We describe a new platform for the positive selection of single live mammalian cells while the cells remain adherent to their growth surface. Cells were grown on arrays of microfabricated, releasable elements composed of SU-8 polymer termed 'cell pallets'. The presence of air between the elements restricted the cells to the top surfaces of the pallets. Single pallets situated within large arrays of pallets were released on demand using a single, focused, laser pulse. The laser pulses were low in energy (2-5 [micro]J) and did not detach nearby, nontargeted pallets. Since the SU-8 pallets and the underlying glass substrate were optically transparent, the cells on the pallets could be visualized by microscopy before and after release. Over 90% of cells remained attached to the pallet during laser-based release. The feasibility of growing the cells from the released pallets into clonal colonies was demonstrated. The pallet array system permits adherent cells to be inspected using conventional microscopy and selected cells released for further analysis. The ability to assess cells while they remain adherent to a surface will broaden the number of attributes that can be utilized for cell separation, for example, cell shape, cytoskeletal properties, and other attributes.

A new photografting method to micropattern a covalent surface modification on poly(dimethylsiloxane) (PDMS) provides advantages in simplicity and efficiency. To accomplish the entire process on the benchtop, the PDMS was initially treated with benzophenone dissolved in a water/acetone mixture. This process permitted limited diffusion of the photoinitiator into the PDMS surface. Polymerization of acrylic acid was initiated by exposure of the benzophenone-implanted PDMS to UV radiation through a photomask with a thin aqueous layer of acrylic acid sandwiched between the PDMS and photomask. This procedure resulted in patterned poly(acrylic acid) (PAA) on the PDMS surface. In the modified regions, PAA and PDMS formed an interpenetrating polymer network extending 50 [micro]m into the PDMS with an X-Y spatial resolution of 5 [micro]m. The carboxyl groups of the PAA graft could be derivatized to covalently bond other molecules to the patterned PAA. Two bioanalytical applications of this micropatterned surface were demonstrated: (1) a guide for cell attachment and growth and (2) a substrate for immunoassays. 3T3 cells were shown to selectively localize to modified surface regions where they could be cultured for up to 7 days. Additionally, the micro-patterned surface was used to immobilize either protein A or antibody for heterogeneous immunoassays.

Alveolar bone defect reconstruction is a common challenge in stomatology. To address this, a thermosensitive/photosensitive gelatin methacrylate (GelMA) gel was developed based on various air solubilities and light-curing technologies. The gel was synthesized by using a freeze-ultraviolet (FUV) method to form a porous and quickly (within 15 min) solidifying modified network structure. Unlike other gel scaffolds limited by complex preparation procedures and residual products, this FUV-GelMA gel shows favorable manufacturing ability, promising biocompatibility, and adjustable macroporous structures. The results from a rat model suggested that this gel scaffold creates a conducive microenvironment for mandible reconstruction and vascularization. In vitro experiments further confirmed that the FUV-GelMA gel promotes osteogenic differentiation of human bone marrow mesenchymal stem cells and angiogenesis of human umbilical vein endothelial cells. Investigation of the underlying mechanism focused on the p38 mitogen-activated protein kinase (MAPK) pathway. We found that SB203580, a specific inhibitor of p38 MAPK, abolished the therapeutic effects of the FUV-GelMA gel on osteogenesis and angiogenesis, both in vitro and in vivo. These findings introduced a novel approach for scaffold-based tissue regeneration in future clinical applications.

Conventional drug delivery systems for natural clay materials still face critical challenges in their practical application, including multiple bacterial infections, combined infection of bacteria and fungi, and low sterilization efficiency. In this work, we address these challenges using the multifunctional montmorillonite nanosheet-based (MMT-based) drug nanoplatform, which involves the antibiotic 5-fluorocytosine (5-FC), antibacterial metal copper ions, and quaternized chitosan (QCS). Composite material QCS/MMT/5-FCCu can can strongly inhibit Staphylococcus aureus (a typical Gram-positive bacterium), Escherichia coli (a typical Gram-negative bacterium), and Candida albicans (a fungus) because 5-FC coordinates with copper ions in situ and due to the deposition of QCS. The subsequent drug release behavior of 5-FCCu was studied, and the results show an initial high concentration kills microorganisms and long-acting sustained release inhibition. Moreover, in vivo wound experiments and toxicity experiments show the promotion of wound healing and excellent biocompatibility. As a demonstration of the utility of the latter, we have shown that the MMT-based smart platform can be used for the treatment of mixed infections of wounds.

Organoid culture has had a significant impact on in vitro studies of the intestinal epithelium; however, the exquisite architecture, luminal accessibility, and lineage compartmentalization found in vivo has not been recapitulated in the organoid systems. We have used a microengineered platform with suitable extracellular matrix contacts and stiffness to generate a self-renewing mouse colonic epithelium that replicates key architectural and physiological functions found in vivo , including a surface lined with polarized crypts. Chemical gradients applied to the basal-luminal axis compartmentalized the stem/progenitor cells and promoted appropriate lineage differentiation along the in vitro crypt axis so that the tissue possessed a crypt stem cell niche as well as a layer of differentiated cells covering the luminal surface. This new approach combining microengineered scaffolds, native chemical gradients, and biophysical cues to control primary epithelium ex vivo can serve as a highly functional and physiologically relevant in vitro tissue model., Competing Interests: Notes The authors declare the following competing financial interest(s): N.L.A., Y.W., C.E.S., S.T.M., and S.J.B. have a financial interest in Altis Biosystems LLC.

Mutant epidermal growth factor receptor (EGFR) is a major driver of non-small-cell lung cancer (NSCLC). Marketed first generation inhibitors, such as erlotinib, effect a transient beneficial response in EGFR mutant NSCLC patients before resistance mechanisms render these inhibitors ineffective. Secondary oncogenic EGFR mutations account for approximately 50% of relapses, the most common being the gatekeeper T790M substitution that renders existing therapies ineffective. The discovery of PF-06459988 (1), an irreversible pyrrolopyrimidine inhibitor of EGFR T790M mutants, was recently disclosed.1 Herein, we describe our continued efforts to achieve potency across EGFR oncogenic mutations and improved kinome selectivity, resulting in the discovery of clinical candidate PF-06747775 (21), which provides potent EGFR activity against the four common mutants (exon 19 deletion (Del), L858R, and double mutants T790M/L858R and T790M/Del), selectivity over wild-type EGFR, and desirable ADME properties. Compound 21 is currently being evaluated in phase-I clinical trials of mutant EGFR driven NSCLC.

Microraft arrays were developed to select and separate cells based on a complex phenotype, weak intercellular adhesion, without knowledge of cell-surface markers or intracellular proteins. Since the cells were also not competent to bind to a culture surface, a method to encapsulate nonadherent cells within a gelatin plug on the concave microraft surface was developed, enabling release and collection of the cells without the need for cell attachment to the microraft surface. After microraft collection, the gelatin was liquified to release the cell(s) for culture or analysis. A semiautomated release and collection device for the microrafts demonstrated 100 ± 0% collection efficiency of the microraft while increasing throughput 5-fold relative to that of manual release and collection. Using the microraft array platform along with the gelatin encapsulation method, single cells that were not surface-attached were isolated with a 100 ± 0% efficiency and a 96 ± 4% postsort single-cell cloning efficiency. As a demonstration, Epstein-Barr virus-infected lymphoblastoid cell lines (EBV-LCL) were isolated based on their intercellular adhesive properties. The identified cell colonies were collected with a 100 ± 0% sorting efficiency and a postsort viability of 87 ± 3%. When gene expression analysis of the EBV latency-associated gene, EBNA-2, was performed, there was no difference in expression between blasting or weakly adhesive cells and nonblasting or nonadhesive cells. Microraft arrays are a versatile method enabling separation of cells based on complicated and as yet poorly understood cell phenotypes.

Glucagon-like peptide-1 (GLP-1) possesses multiple physiological functions, which make it a potential drug candidate for the treatment of type 2 diabetes. However, its clinical application was limited severely by its short half-life in vivo. Therefore, stabilization of GLP-1 is critical for the use of this peptide in drug development. In this study, a novel GLP-1 derivative, VGLP1K6, processed a significantly prolonged half-life in vivo. Structural analysis using molecular dynamics simulations demonstrated that VGLP1K6 has a rigid V-shaped conformation resulting from the intrapeptide disulfide bond. The C-terminal polylysine residues of VGLP1K6 caused the vulnerable N-terminus of GLP-1 (HA-fragment) to reside within the pocket-like cavity of the peptide due to the intrahydrogen bonds. The structural analysis suggested that this structural alteration contributed to the remarkable prolonged half-life of VGLP1K6, which was approximately 70 h. In addition, VGLP1K6 induced better long-acting glucose tolerance and greater HbA1c reductions than GLP-1 in rodents. Our findings suggest that the GLP-1 derivative VGLP1K6 might be a possible potent antidiabetic drug for the treatment of type 2 diabetes mellitus.

Three-carbon axial chirality has been asymmetrically established from racemic one-carbon central chirality efficiently at room temperature: we report here the discovery of the first catalytic asymmetric carbonylation of readily available racemic propargylic carbonates to access optically active 2,3-allenoates with fairly high ee. The combination of [(π-allyl)PdCl]2 with [(R)-ECNU-Phos], a new chiral bisphosphine ligand based on a biphenyl skeleton, demonstrates high enantioselectivity. Both enantiomers of allenoates can be obtained at room temperature by applying either (R)- or (S)-ECNU-Phos.

An efficient bimetallic Zn(II)/Cu(I)-mediated asymmetric synthesis of simple axially chiral allenes from terminal alkynes and aldehydes was realized by taking advantage of the chiral amine (S)-α,α-diphenylprolinol 3. This one-pot procedure is compatible with broad scopes of both terminal alkynes and aldehydes, providing axially chiral allenes in practical yields with an excellent enantioselectivity. Control experiments revealed that CuBr is responsible for the efficient formation of propargylic amine while the combination of CuBr and ZnBr2 plays crucial roles in the amine-to-allene transformation.

We report on the synthesis and characterization of end-tethering polypeptide monolayers based on poly(beta-benzyl-L-aspartate) (PBLA) homopolymer and PBLA-b-poly(gamma-benzyl-L-glutamate) block copolymer. The homopolypeptide and copolypeptide brushes were fabricated by the sequential, surface-initiated vapor deposition polymerization of the N-carboxyanhydride of beta-benzyl-L-aspartate or gamma-benzyl-L-glutamate, yielding 80-nm-thick, chemically grafted films after 30 min of reaction time. Both Fourier transform infrared spectrometry and circular dichroism showed that the polypeptide brushes could be reversibly and repeatedly switched between left-handed and right-handed alpha-helical structures in response to solvent vapor exposure or permanently converted to a beta-sheet structure when heated to 160 degrees C in air. The facile, in vacuo manufacturability and the robustness of the films of PBLA-based brushes could allow them to be incorporated as active components for biosensing and nanofabricated devices.

End-tethered cationic polypeptide brushes of poly(L-lysine) (t-PLL) were combined with three anionic polymers, poly(acrylic acid) (PAA), poly(L-glutamic acid) (PLGA), and poly(L-aspartic acid) (PLAA), to form reversible polyelectrolyte complex films at surfaces at neutral pH. The polyelectrolyte complex formation was confirmed by an in situ zeta-potential study and by positive fluorescent images after adding prelabeled anionic polymers. The secondary conformations of the t-PLL complex films depend upon the specific polyelectrolyte with which t-PLL was coupled as studied by circular dichroism and FTIR. Specifically, the random coil chain configuration of the t-PLL film was converted to an alpha-helical, beta-sheet, or random coil structure after forming complexes with PAA, PLGA, or PLAA, respectively. Each of these complexes could be returned to the original random coil t-PLL structure by a dilute acid rinse. Additional thickness and morphological studies from ellipsometry and atomic force microscopy have further shown that the corresponding film thicknesses of the individual solvated films were affected more by the secondary structures in films than by the adsorbed mass or surface net charges. The solvated thickness was reduced significantly after the random coil t-PLL film was coupled with polyanions in forming compact regulated structures in films. This biomimetic approach provides a new opportunity for controlling the molecular organization in surface macromolecular assemblies and may provide a model for structural study of protein complexes on a chip.

We report a simple approach to fabricate heterogeneously wetted surfaces which can be used to pattern living cells or biomolecules. An array of pedestals composed of SU-8 was fabricated on a glass surface which was then derivatized with a hydrophobic silane. Upon addition of aqueous solutions to the array, air was trapped within the hydrophobic cavities between the pedestals. The trapped air formed a "virtual wall" blocking access to these cavities. Cells cultured on the array were forced to grow only on the tops of the pedestals, i.e., the surfaces wetted by aqueous media. The virtual walls were stable during manipulation of the array and over long time periods (months).

SU-8 has gained widespread acceptance as a negative photoresist. It is also finding increasing use as a structural material in microanalytical devices. Consequently, methods to tailor the surface properties of SU-8 as well as to micropattern coatings on the surface of SU-8 are needed. The SU-8 photoresist consists of EPON SU-8 resin mixed with the photoacid generator triarylsulfonium hexafluoroantimonate. This photoacid generator can also serve as a photoinitiator generating free radicals when illuminated with UV light. Under the appropriate conditions, sufficient triarylsulfonium hexafluoroantimonate remains within cured SU-8 to act as a source of free radicals and initiate UV-mediated grafting of polymers onto the surface of the SU-8. UV-mediated grafting was used to coat SU-8 surfaces with poly(acrylic acid) and other water-soluble monomers. The SU-8 surface was chemically micropatterned by placing a mask between the UV light and SU-8. The X-Y spatial resolution of micropatterned poly(acrylic acid) on the SU-8 surface was 2 mum. Three applications of these chemically modified SU-8 surfaces were demonstrated. In the first, poly(ethylene glycol) was used to protect the SU-8 from interactions with proteins, yielding a surface resistant to biofouling. In the second demonstration, the SU-8 surface was micropatterned with a cell-resistant layer to guide cellular attachment and growth. In the final application, SU-8 micropallets were encoded with polymer lines. The bar codes were read by either absorbance or fluorescence measurements. Thus, UV-mediated graft polymerization is an efficient and effective method to micropattern coatings onto the surface of SU-8.

A simple "solvent quenching" approach to align an alpha-helical end-grafted poly(gamma-benzyl l-glutamate) monolayer is presented. By sequentially treating poly(gamma-benzyl l-glutamate) with good solvent (chloroform) and bad solvent (acetone), we obtained a highly aligned monolayer with an average tilt angle as small as 3 degrees .