Microfluidics-assisted fabrication of natural killer cell-laden microgel enhances the therapeutic efficacy for tumor immunotherapy.

Recently, interest in cancer immunotherapy has increased over traditional anti-cancer therapies such as chemotherapy or targeted therapy. Natural killer (NK) cells are part of the immune cell family and essential to tumor immunotherapy as they detect and kill cancer cells. However, the disadvantage of NK cells is that cell culture is difficult. In this study, porous microgels have been fabricated using microfluidic channels to effectively culture NK cells. Microgel fabrication using microfluidics can be mass-produced in a short time and can be made in a uniform size. Microgels consist of photo cross-linkable polymers such as methacrylic gelatin (GelMa) and can be regulated via controlled GelMa concentrations. NK92 cell-laden three-dimensional (3D) microgels increase mRNA expression levels, NK92 cell proliferation, cytokine release, and anti-tumor efficacy, compared with two-dimensional (2D) cultures. In addition, the study confirms that 3D-cultured NK92 cells enhance anti-tumor effects compared with enhancement by 2D-cultured NK92 cells in the K562 leukemia mouse model. Microgels containing healthy NK cells are designed to completely degrade after 5 days allowing NK cells to be released to achieve cell-to-cell interaction with cancer cells. Overall, this microgel system provides a new cell culture platform for the effective culturing of NK cells and a new strategy for developing immune cell therapy.

NK cells encapsulated in micro/macropore-forming hydrogels via 3D bioprinting for tumor immunotherapy.

BACKGROUND: Patients face a serious threat if a solid tumor leaves behind partial residuals or cannot be completely removed after surgical resection. Immunotherapy has attracted attention as a method to prevent this condition. However, the conventional immunotherapy method targeting solid tumors, that is, intravenous injection, has limitations in homing in on the tumor and in vivo expansion and has not shown effective clinical results. METHOD: To overcome these limitations, NK cells (Natural killer cells) were encapsulated in micro/macropore-forming hydrogels using 3D bioprinting to target solid tumors. Sodium alginate and gelatin were used to prepare micro-macroporous hydrogels. The gelatin contained in the alginate hydrogel was removed because of the thermal sensitivity of the gelatin, which can generate interconnected micropores where the gelatin was released. Therefore, macropores can be formed through bioprinting and micropores can be formed using thermally sensitive gelatin to make macroporous hydrogels. RESULTS: It was confirmed that intentionally formed micropores could help NK cells to aggregate easily, which enhances cell viability, lysis activity, and cytokine release. Macropores can be formed using 3D bioprinting, which enables NK cells to receive the essential elements. We also characterized the functionality of NK 92 and zEGFR-CAR-NK cells in the pore-forming hydrogel. The antitumor effects on leukemia and solid tumors were investigated using an in vitro model. CONCLUSION: We demonstrated that the hydrogel encapsulating NK cells created an appropriate micro-macro environment for clinical applications of NK cell therapy for both leukemia and solid tumors via 3D bioprinting. 3D bioprinting makes macro-scale clinical applications possible, and the automatic process shows potential for development as an off-the-shelf immunotherapy product. This immunotherapy system could provide a clinical option for preventing tumor relapse and metastasis after tumor resection. Micro/macropore-forming hydrogel with NK cells fabricated by 3D bioprinting and implanted into the tumor site.

Microfluidic device for one-step detection of breast cancer-derived exosomal mRNA in blood using signal-amplifiable 3D nanostructure.

Metastasis attributed to approximately 90% of cancer-related deaths; hence, the detection of metastatic tumor-derived components in the blood assists in determining cancer recurrence and patient survival. Microfluidic-based sensors facilitate analysis of small fluid volumes and represent an accurate, rapid, and user-friendly method of field diagnoses. In this study, we have developed a microfluidic chip-based exosomal mRNA sensor (exoNA-sensing chip) for the one-step detection of exosomal ERBB2 in the blood by integrating a microfluidic chip and 3D-nanostructured hydrogels. The exoNA-sensing chip is a vacuum-driven power-free microfluidic chip that can accurately control the flow of trace fluids (<100 µL). The sensing part of the exoNA-sensing chip includes 3D-nanostructured hydrogels capable of detecting ERBB2 and a reference gene by amplifying a fluorescent signal via an enzyme-free catalytic hairpin assembly reaction at room temperature. This hydrogel offers a detection limit of 58.3 fM with good selectivity for target sequences. The performance of the exoNA-sensing chip was evaluated by testing in vitro and in vivo samples and was proven to be effective for cancer diagnosis and liquid biopsies.

Flexible nanopillar-based immunoelectrochemical biosensor for noninvasive detection of Amyloid beta.

The noninvasive early detection of biomarkers for Alzheimer's disease (AD) is essential for the development of specific treatment strategies. This paper proposes an advanced method for fabricating highly ordered and flexible nanopillar-based electrochemical biosensors by the combination of soft/photolithography and metal evaporation. The nanopillar array (NPA) exhibits high surface area containing 1500 nm height and 500 nm diameter with 3:1 ratio. In regard with physical properties of polyurethane (PU) substrate, the developed NPA is sustainable and durable to external pressure such as bending and twisting. To manipulate the NPA surface to biocompatible, the gold was uniformly deposited on the PU substrate. The thiol chemistry which is stably modified on the gold surface as a form of self-assembled monolayer was employed for fabricating the NPA as a biocompatible chip by covalently immobilize the antibodies. The proposed nanopillar-based immunoelectrochemical biosensor exhibited good and stable electrochemical performance in ß-amyloid (Aß) detection. Moreover, we successfully confirmed the performance of the as-developed sensor using the artificial injection of Aß in human tear, with sensitivity of 0.14 ng/mL and high reproducibility (as a standard deviation below 10%). Our findings show that the developed nanopillar-based sensor exhibits reliable electrochemical characteristics and prove its potential for application as a biosensor platform for testing at the point of care.

Peptidoglycan-Binding Protein Metamaterials Mediated Enhanced and Selective Capturing of Gram-Positive Bacteria and Their Specific, Ultra-Sensitive, and Reproducible Detection via Surface-Enhanced Raman Scattering.

Biological metamaterials with a specific size and spacing are necessary for developing highly sensitive and selective sensing systems to detect hazardous bacteria in complex solutions. Herein, the construction of peptidoglycan-binding protein (PGBP)-based metamaterials to selectively capture Gram-positive cells with high efficacy is reported. Nanoimprint lithography was used to generate a nanohole pattern as a template, the inside of which was modified with nickel(II)-nitrilotriacetic acid (Ni-NTA). Then, PGBP metamaterials were fabricated by immobilizing PGBP via chelation between Ni-NTA and six histidines on PGBP. Compared to the flat and spread PGBP-covered bare substrates, the PGBP-based metamaterials enabled selective capturing of Gram-positive bacteria with high efficacy, owing to enhanced interactions between the metamaterials and bacterial surface not shown in bulk materials. Thereafter, the specific strain and quantitative information of the captured bacteria was obtained by surface-enhanced Raman scattering mapping analysis in the 1 to 1 × 106 cfu/mL range within 30 min. It should be noted that no additional signal amplification process was required for lowly abundant bacteria, even at the single-bacterium level. The PGBP-based metamaterials could be regenerated multiple times with preserved sensing efficiency. Finally, this assay can detect specific Gram-positive bacteria, such as Staphylococcus aureus, in human plasma.

Antibacterial Nanopillar Array for an Implantable Intraocular Lens.

Postsurgical intraocular lens (IOL) infection caused by pathogenic bacteria can result in blindness and often requires a secondary operation to replace the contaminated lens. The incorporation of an antibacterial property onto the IOL surface can prevent bacterial infection and postoperative endophthalmitis. This study describes a polymeric nanopillar array (NPA) integrated onto an IOL, which captures and eradicates the bacteria by rupturing the bacterial membrane. This is accomplished by changing the behavior of the elastic nanopillars using bending, restoration, and antibacterial surface modification. The combination of the polymer coating and NPA dimensions can decrease the adhesivity of corneal endothelial cells and posterior capsule opacification without causing cytotoxicity. An ionic antibacterial polymer layer is introduced onto an NPA using an initiated chemical vapor deposition process. This improves bacterial membrane rupture efficiency by increasing the interactions between the bacteria and nanopillars and damages the bacterial membrane using quaternary ammonium compounds. The newly developed ionic polymer-coated NPA exceeds 99% antibacterial efficiency against Staphylococcus aureus, which is achieved through topological and physicochemical surface modification. Thus, this paper provides a novel, efficient strategy to prevent postoperative complications related to bacteria contamination of IOL after cataract surgery.

The role of hydrophobic silane coating on Si stamps in nanoimprint lithography.

Hydrophobic silane coatings have been successfully applied to the surface of Si stamps to improve demolding in nanoimprint lithography (NIL). However, the role of the silane coating has only been studied either indirectly, by measuring adhesion or friction coefficients for Si and substrate surfaces without patterns, or collectively, by measuring the overall demolding force that does not differentiate contributions of friction dissipation, stored elastic energy, and adhesion. Here, for the first time, we present experimental evidence on the role of the silane coating in improving demolding in UV-NIL by using different silane coatings. The silane coatings were characterized by x-ray photoelectron spectroscopy, water contact angle, and friction force measurements. Then, the work of demolding was systematically measured for different silane coatings using stamps with the same micropattern but different pattern depths. Comparison of the results to the theoretical model developed for fiber-matrix debonding energy by Sutcu and Hillig [Acta Metall. Mater. 38(12), 2653-2662] indicated that with a hydrophobic silane coating, the main parameter contributing to overall demolding work shifts from adhesion to stored elastic energy and frictional dissipation as surface adhesion keeps decreasing. The results confirm that the main role of the silane coating in reducing the demolding is to reduce surface adhesion rather than friction at the stamp/substrate interface.

X Y parallel compliant stage with compact configuration.

This paper presents a piezo-driven compliant stage for nano positioning with two degree-of-freedom parallel linear motions. Nano positioning is one of the most important factors in completion of nanotechnologies. It can be accomplished by flexure-based compliant stages driven by piezo-actuators. For compact configuration, the compliant stage is stacked by two-layered compliant mechanisms. The upper layer contains a motion guide mechanism, and the lower layer two displacement amplification mechanisms. The motion guide mechanism consists of four prismatic-prismatic parallel compliant joint chains for two translational motion guides. The displacement amplification mechanism is adopted by a flexure-based flextensional amplification mechanism driven by a stack-type piezo actuator. Due to the parallel compliant joint chains, the stage has the same dynamics in the x and y axes. In this study, through design and analysis, the mechanisms were machined via wire electro-discharge machining and are were then integrated with two stack-type piezoelectric elements for actuation, and two capacitive sensors for ultra-precision displacement measurement. Finally, experiments were carried out to demonstrate the performance of the compliant stage.

Fabrication of an inorganic nano structure for a large area via electrohydrodynamic lithography (EHL).

Electrohydrodynamic lithography (EHL) is one of several unusual lithographic techniques for fabricating sub-micrometer structures over a large area. EHL uses the electrohydrodynamic (EHD) film instability induced by a laterally modulated electric field, which allows it to fabricate not only general organic structures but also structures of various components, such as diblock copolymers and inorganic materials, without contact between the resist and the stamp. Furthermore, EHL is a very special lithographic technique in that diverse structures are fabricated from one stamp via electric field modulation. The electric field is controlled by the replication time, air layer thickness, etc. A replicated inorganic structure was demonstrated from a hexagonal hole and line arrays. The heat treatment of the replicated pattern was carried out to obtain the crystalline phase, after which the samples were characterized via Raman spectroscopy. These values were ascertained using field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM). The results will be useful in providing a facile route for patterning functional metal oxides over a large area. Such a technique can be used to produce photovoltaic cells, memory devices, display devices, etc.

Replication of a thin polydimethylsiloxane stamp and its application to dual-nanoimprint lithography for 3D hybrid nano/micropatterns.

This paper presents the fabrication of a thin and flexible polydimethylsiloxane (PDMS) stamp with a thickness of a few tens of um and its application to nanoimprint lithography (NIL). The PDMS material generally has a low elastic modulus and high adhesive characteristics. Therefore, after being treated, the thin PDMS stamp is easily deformed and torn, adhering to itself and other materials. This paper introduces the use of a metal ring around the flange of a thin PDMS stamp to assist with the handling of this material. A PDMS stamp with a motheye pattern in nanometer scale was inserted between a substrate and a microstamp with concave patterns in micrometer scale. Subsequently, three-dimensional (3D) hybrid nano/micropatterns were fabricated by pressing these two stamps and curing the resist. The fabricated hybrid patterns were measured and verified in both the microscale and nanoscale. The process, termed "dual NIL," can be applied to the fabrication of optical components or bio-sensors that require repetitive nanopatterns on micropatterns.

A simulation study on the effect of cross-linking agent concentration for defect tolerant demolding in UV nanoimprint lithography.

The chemistry and composition of UV-sensitive resists are key factors determining the stress in the molded resist structure in UV nanoimprint lithography (UV-NIL) and thus the success of the process. The stress in the molded structure is mainly generated due to shrinkage of the resist in the UV curing step and also adhesion and friction at the stamp/resist interface in the subsequent demolding step. Thus, understanding of the stress generated in these steps is critical to the improvement of the process as well as the development of new UV resists. In this paper the effect of resist composition on the stress generation was studied by numerical simulations of the curing and demolding steps in UV-NIL. Parameters required for the simulation, such as resist shrinkage, Young's modulus, fracture strength, friction coefficient, crack initiation stress, and debonding energy, were determined experimentally for different resist compositions. As the cross-linking agent concentration increases the fracture strength also improves. In addition, as more cross-linking agent is added to the resist composition, both shrinkage stress due to the curing and also adhesion at the stamp/resist interface increase resulting in a larger maximum local stress experienced by the resist on demolding. By normalizing the overall maximum local stress by the fracture stress of the resist, we found that there is an optimum for the cross-linking agent concentration that leads to the most successful imprinting. Our finding is also corroborated by qualitative experimentations performed for UV-NIL with various resist compositions.

Highly sensitive biosensing using arrays of plasmonic Au nanodisks realized by nanoimprint lithography.

We describe the fabrication of elliptical Au nanodisk arrays as a localized surface plasmon resonance (LSPR) sensing substrate for clinical immunoassay via thermal nanoimprint lithography (NIL) and enhancement in the sensitivity of the detection of the prostate-specific antigen (PSA) using the precipitation of 5-bromo-4-chloro-3-indolyl phosphate p-toluidine/nitro blue tetrazolium (BCIP/NBT), catalyzed by alkaline phosphatase. Au nanodisks were fabricated on glass through an unconventional tilted evaporation, which could preserve the thickness of imprinted resists and create an undercut beneficial to the subsequent lift-off process without any damage to pattern dimension and the glass while removing the residual polymers. To investigate the optically anisotropic property of the LSPR sensors, a probe light with linear polarization parallel to and perpendicular to the long axis of the elliptical nanodisk array was utilized, and their sensitivity to the bulk refractive index (RI) was measured as 327 and 167 nm/RIU, respectively. To our knowledge, this is the first application of enzyme-substrate reaction to sandwich immunoassay-based LSPR biosensors that previously suffered from a low sensitivity due to the short penetration depth of the plasmon field, especially when large-sized antibodies were used as bioreceptors. As a result, a large change in local refractive index because of the precipitation on the Au nanodisks amplified the wavelength shift of the LSPR peak in the vis-NIR spectrum, resulting in femtomolar detection limits, which was ∼10(5)-fold lower than the label-free detection without the enzyme precipitation. This method can be extended easily to the other clinical diagnostics with a high sensitivity.

Nano-sized silver patterning using nano-ink with an indirect patterning method of nanoimprint and lift-off processes.

Silver nanopatterns were fabricated using a nanoimprint and lift-off process using silver nano-ink. Particle-based silver nano-ink was used to fabricate metal patterns below 100 nm in a lift-off process without the undercut shape of polymer patterns typically required in a conventional lift-off process using evaporated metal. The silver film made by spin-coating the silver nano-ink and removing the solvent using PDMS, has a low density compared to evaporated metal film; hence, unnecessary nano-ink that is not part of the pattern area is easily eliminated during the lift-off process. Using the proposed patterning process, patterns defined on a stamp were successfully transferred to the nano-ink through nanoimprinted polymer patterns on a four-inch Si wafer. Compared to other metal patterning methods that involve lift-off processes, the proposed metal patterning process is a simple and cost-effective process capable of fabricating the micro- and nano-sized metal patterns due to its low operating temperature and one-step polymer patterning process by nanoimprinting.

Enhanced biomolecular detection based on localized surface plasmon resonance (LSPR) using enzyme-precipitation reaction.

An enzyme-catalyzed precipitation reaction was employed as a means to increase the change in the LSPR signal after intermolecular bindings between antigens and antibodies occurred on gold nanodot surfaces. The gold nanodot array with an diameter of 175 nm and a thickness of 20 nm was fabricated on a glass wafer using thermal nanoimprint lithography. The human interleukin (hIL) 5 antibody was immobilized on the gold nanodot, followed by binding of hIL 5 to the anti-hIL 5. Subsequently, a biotinylated anti-hIL 5 and a alkaline phosphatase conjugated with streptavidin were simultaneously introduced. A mixture of 5-bromo-4-chloro-3-indolyl phosphate p-toluidine (BCIP) and nitro blue tetrazolium (NBT) was then used for precipitation, which resulted from the biocatalytic reaction of the alkaline phosphatase on gold nanodot. The LSPR spectra were obtained after each binding process. Using this analysis, the enzyme-catalyzed precipitation reaction on gold nanodots was found to be effective in amplifying the change in the peak wavelength of LSPR after molecular bindings.

The fabrication scheme of a high resolution and high aspect ratio UV-nanoimprint mold.

We propose a new scheme of fabricating molds for UV-nanoimprint lithography (UV-NIL) that is both high resolution and has a high aspect ratio. The scheme involves the utilization of a hydrogen silsesquioxane (HSQ) electron beam resist for high resolution patterning and the sputter-deposited alpha-Si layer that defines the high-aspect-ratio mold pattern obtained from the high etch selectivity between the HSQ and the alpha-Si. We obtained high resolution line patterns and dot patterns with feature sizes of 40 nm and 25 nm, respectively. The aspect ratio of the patterns was about 3.5 for line patterns and about 5 for dot patterns. These molds also demonstrate successful UV-nanoimprint patterning.

Adhesion force measurement between the stamp and the resin in ultraviolet nanoimprint lithography--an investigative approach.

In the ultraviolet nanoimprint lithography (UV-NIL) process, the surface interaction between the mold and the resist is essential along with molecularly clean separation of the mold from the surface of the cured resist for repeated use of the mold. In our present study, various mold-resin combinations have been examined by a tensile strength method to determine the adhesion force between a mold with a relief pattern and a photocurable resin. The adhesion force of polymer molds of the fluorine-containing polymers perfluoropolyether dimethacrylate (PFPE) and polydimethylsiloxane (PDMS) were compared to those of hard molds such as Si and quartz against several commercial UV-NIL resins. Eventually, PFPE with higher molecular weight-AMONIL-was found to be an excellent UV-NIL mold-resin combination with the lowest adhesion force per unit area (20 kPa). In particular, the 36-fold repeated imprinting with a high molecular weight PFPE mold showed only a slight increase of adhesion force by 36 kPa with molecularly clean release from the AMONIL resin, whereas a surface-modified PDMS mold revealed highly increased adhesion from an initial 20 kPa to 120 kPa after repeated use.

Electron beam lithography-assisted fabrication of Au nano-dot array as a substrate of a correlated AFM and confocal Raman spectroscopy.

This paper documents a study of an Au nano-dot array that was fabricated by electron beam lithography on a glass wafer. The patterns that had features of 100 nm dots in diameter with a 2-mum pitch comprised a total area of 200 x 200 microm(2). The dot-shaped Cr underlayer was open to the air after developing Poly(methyl methacrylate) (PMMA). When dipped into the Cr etchant, the exposed Cr layer was eliminated from the glass wafer in a short period of time. In order to ultimately fabricate the Ti/Au dot arrays, Ti and Au were deposited onto the arrays with a thickness of 2 and 40 nm, respectively. The lift-off procedure was carried out in the Cr etchant using sonication in order to completely remove the residual Cr/PMMA layer. The fabricated Au nano-dot array was then immersed in an Ag enhancing solution and then into an ethanol solution containing (N-(6-(Biotinamido)hexyl)-3'-(2'-pyridyldithio)-propionamide (Biotin-HPDP). The substrate was analyzed using a correlated atomic force microscopy (AFM) and confocal Raman spectroscopy. Through this procedure, position-dependent surface-enhanced Raman spectroscopy (SERS) signals could be obtained.

Fluoropolymer synthesis and its application as a mold material in UV-nano-imprint lithography process.

In our present work, we report the synthesis of a novel, highly photosensitive photocurable methacrylate functionalized perfluoropolyether (fluoropolymer) by grafting methacrylate functional group onto the backbone of perfluoropolyether chain. Reaction mechanism for the synthesis of the oligomer has been clearly described in this article. Dihydroxy terminated perfluoropolyether (PFPE-diol) monomer was made photocurable by carrying out its reaction with ethyl isocyanato methacrylate (EIM) by a simple addition reaction. The product was characterized by NMR and FT-IR analysis. The synthesized polymeric material has potential application in soft lithographic process, as a viable option for PDMS (polydimethylsiloxane). Mold materials made from fluoropolymer have higher modulus, when compared to PDMS and posses better pattern relief property than rigid materials such as, quartz. In particular this fluoropolymer can be used as stamping material in nano fabrication, especially in UV-based Nano-imprinting lithographic (NIL) process.

Enhanced ionic conductivity of AgI nanowires/AAO composites fabricated by a simple approach.

AgI nanowires/anodic aluminum oxide (AgI NWs/AAO) composites have been fabricated by a simple approach, which involves the thermal melting of AgI powders on the surface of the AAO membrane, followed by the infiltration of the molten AgI inside the nanochannels. As-prepared AgI nanowires have corrugated outer surfaces and are polycrystalline according to scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations. X-ray diffraction (XRD) shows that a considerable amount of 7H polytype AgI exists in the composites, which is supposed to arise from the interfacial interactions between the embedded AgI and the alumina. AC conductivity measurements for the AgI nanowires/AAO composites exhibit a notable conductivity enhancement by three orders of magnitude at room temperature compared with that of pristine bulk AgI. Furthermore, a large conductivity hysteresis and abnormal conductivity transitions were observed in the temperature-dependent conductivity measurements, from which an ionic conductivity as high as 8.0 × 10(2) Ω(-1) cm(-1) was obtained at around 70 °C upon cooling. The differential scanning calorimetry (DSC) result demonstrates a similar phase transition behavior as that found in the AC conductivity measurements. The enhanced ionic conductivity, as well as the abnormal phase transitions, can be explained in terms of the existence of the highly conducting 7H polytype AgI and the formation of well-defined conduction paths in the composites.