Chemical Lift-Off Lithography of Metal and Semiconductor Surfaces.

Chemical lift-off lithography (CLL) is a subtractive soft-lithographic technique that uses polydimethylsiloxane (PDMS) stamps to pattern self-assembled monolayers of functional molecules for applications ranging from biomolecule patterning to transistor fabrication. A hallmark of CLL is preferential cleavage of Au-Au bonds, as opposed to bonds connecting the molecular layer to the substrate, i.e., Au-S bonds. Herein, we show that CLL can be used more broadly as a technique to pattern a variety of substrates composed of coinage metals (Pt, Pd, Ag, Cu), transition and reactive metals (Ni, Ti, Al), and a semiconductor (Ge) using straightforward alkanethiolate self-assembly chemistry. We demonstrate high-fidelity patterning in terms of precise features over large areas on all surfaces investigated. We use patterned monolayers as chemical resists for wet etching to generate metal microstructures. Substrate atoms, along with alkanethiolates, were removed as a result of lift-off, as previously observed for Au. We demonstrate the formation of PDMS-stamp-supported bimetallic monolayers by performing CLL on two different metal surfaces using the same PDMS stamp. By expanding the scope of the surfaces compatible with CLL, we advance and generalize CLL as a method to pattern a wide range of substrates, as well as to produce supported metal monolayers, both with broad applications in surface and materials science.

Scalable Fabrication of Quasi-One-Dimensional Gold Nanoribbons for Plasmonic Sensing.

Plasmonic nanostructures have a wide range of applications, including chemical and biological sensing. However, the development of techniques to fabricate submicrometer-sized plasmonic structures over large scales remains challenging. We demonstrate a high-throughput, cost-effective approach to fabricate Au nanoribbons via chemical lift-off lithography (CLL). Commercial HD-DVDs were used as large-area templates for CLL. Transparent glass slides were coated with Au/Ti films and functionalized with self-assembled alkanethiolate monolayers. Monolayers were patterned with lines via CLL. The lifted-off, exposed regions of underlying Au were selectively etched into large-area grating-like patterns (200 nm line width; 400 nm pitch; 60 nm height). After removal of the remaining monolayers, a thin In2O3 layer was deposited and the resulting gratings were used as plasmonic sensors. Distinct features in the extinction spectra varied in their responses to refractive index changes in the solution environment with a maximum bulk sensitivity of ∼510 nm/refractive index unit. Sensitivity to local refractive index changes in the near-field was also achieved, as evidenced by real-time tracking of lipid vesicle or protein adsorption. These findings show how CLL provides a simple and economical means to pattern large-area plasmonic nanostructures for applications in optoelectronics and sensing.

Photothermal Intracellular Delivery Using Gold Nanodisk Arrays.

Local heating using pulsed laser-induced photothermal effects on plasmonic nanostructured substrates can be used for intracellular delivery applications. However, the fabrication of plasmonic nanostructured interfaces is hampered by complex nanomanufacturing schemes. Here, we demonstrate the fabrication of large-area plasmonic gold (Au) nanodisk arrays that enable photothermal intracellular delivery of biomolecular cargo at high efficiency. The Au nanodisks (350 nm in diameter) were fabricated using chemical lift-off lithography (CLL). Nanosecond laser pulses were used to excite the plasmonic nanostructures, thereby generating transient pores at the outer membranes of targeted cells that enable the delivery of biomolecules via diffusion. Delivery efficiencies of >98% were achieved using the cell impermeable dye calcein (0.6 kDa) as a model payload, while maintaining cell viabilities at >98%. The highly efficient intracellular delivery approach demonstrated in this work will facilitate translational studies targeting molecular screening and drug testing that bridge laboratory and clinical investigations.

Intracellular Photothermal Delivery for Suspension Cells Using Sharp Nanoscale Tips in Microwells.

Efficient intracellular delivery of biomolecules into cells that grow in suspension is of great interest for biomedical research, such as for applications in cancer immunotherapy. Although tremendous effort has been expended, it remains challenging for existing transfer platforms to deliver materials efficiently into suspension cells. Here, we demonstrate a high-efficiency photothermal delivery approach for suspension cells using sharp nanoscale metal-coated tips positioned at the edge of microwells, which provide controllable membrane disruption for each cell in an array. Self-aligned microfabrication generates a uniform microwell array with three-dimensional nanoscale metallic sharp tip structures. Suspension cells self-position by gravity within each microwell in direct contact with eight sharp tips, where laser-induced cavitation bubbles generate transient pores in the cell membrane to facilitate intracellular delivery of extracellular cargo. A range of cargo sizes were tested on this platform using Ramos suspension B cells with an efficiency of >84% for Calcein green (0.6 kDa) and >45% for FITC-dextran (2000 kDa), with retained viability of >96% and a throughput of >100 000 cells delivered per minute. The bacterial enzyme ß-lactamase (29 kDa) was delivered into Ramos B cells and retained its biological activity, whereas a green fluorescence protein expression plasmid was delivered into Ramos B cells with a transfection efficiency of >58%, and a viability of >89% achieved.

A Microfabricated Sandwiching Assay for Nanoliter and High-Throughput Biomarker Screening.

Cells secrete substances that are essential to the understanding of numerous immunological phenomena and are extensively used in clinical diagnoses. Countless techniques for screening of biomarker secretion in living cells have generated valuable information on cell function and physiology, but low volume and real-time analysis is a bottleneck for a range of approaches. Here, a simple, highly sensitive assay using a high-throughput micropillar and microwell array chip (MIMIC) platform is presented for monitoring of biomarkers secreted by cancer cells. The sensing element is a micropillar array that uses the enzyme-linked immunosorbent assay (ELISA) mechanism to detect captured biomolecules. When integrated with a microwell array where few cells are localized, interleukin 8 (IL-8) secretion can be monitored with nanoliter volume using multiple micropillar arrays. The trend of cell secretions measured using MIMICs matches the results from conventional ELISA well while it requires orders of magnitude less cells and volumes. Moreover, the proposed MIMIC is examined to be used as a drug screening platform by delivering drugs using micropillar arrays in combination with a microfluidic system and then detecting biomolecules from cells as exposed to drugs.

Spin-Dependent Ionization of Chiral Molecular Films.

Spin selectivity in photo-emission from ferromagnetic substrates functionalized with chiral organic films was analyzed by ultraviolet photoelectron spectroscopy at room temperature. Using radiation with photon energy greater than the ionization potential of the adsorbed molecules, photoelectrons were collected that originated from both underlying ferromagnetic substrates and the organic films, with kinetic energies in the range of ca. 0-18 eV. We investigated chiral organic films composed of self-assembled monolayers of α-helical peptides and electrostatically adsorbed films of the protein, bovine serum albumin, with different α-helix and ß-sheet contents. Ultraviolet photoelectron spectral widths were found to depend on substrate magnetization orientation and polarization, which we attribute to helicity-dependent molecular ionization cross sections arising from photoelectron impact, possibly resulting in spin-polarized holes. These interactions between spin-polarized photoelectrons and chiral molecules are physically manifested as differences in the measured photoionization energies of the chiral molecular films. Substrate magnetization-dependent ionization energies and work function values were deconvoluted using surface charge neutralization techniques, permitting the measurement of relative spin-dependent energy barriers to transmission through chiral organic films.

Precision-Guided Nanospears for Targeted and High-Throughput Intracellular Gene Delivery.

An efficient nonviral platform for high-throughput and subcellular precision targeted intracellular delivery of nucleic acids in cell culture based on magnetic nanospears is reported. These magnetic nanospears are made of Au/Ni/Si (∼5 µm in length with tip diameters <50 nm) and fabricated by nanosphere lithography and metal deposition. A magnet is used to direct the mechanical motion of a single nanospear, enabling precise control of position and three-dimensional rotation. These nanospears were further functionalized with enhanced green fluorescent protein (eGFP)-expression plasmids via a layer-by-layer approach before release from the underlying silicon substrate. Plasmid functionalized nanospears are guided magnetically to approach target adherent U87 glioblastoma cells, penetrating the cell membrane to enable intracellular delivery of the plasmid cargo. After 24 h, the target cell expresses green fluorescence indicating successful transfection. This nanospear-mediated transfection is readily scalable for the simultaneous manipulation of multiple cells using a rotating magnet. Cell viability >90% and transfection rates >80% were achieved, which exceed conventional nonviral intracellular methods. This approach is compatible with good manufacturing practices, circumventing barriers to the translation and clinical deployment of emerging cellular therapies.

Polymer-Pen Chemical Lift-Off Lithography.

We designed and fabricated large arrays of polymer pens having sub-20 nm tips to perform chemical lift-off lithography (CLL). As such, we developed a hybrid patterning strategy called polymer-pen chemical lift-off lithography (PPCLL). We demonstrated PPCLL patterning using pyramidal and v-shaped polymer-pen arrays. Associated simulations revealed a nanometer-scale quadratic relationship between contact line widths of the polymer pens and two other variables: polymer-pen base line widths and vertical compression distances. We devised a stamp support system consisting of interspersed arrays of flat-tipped polymer pens that are taller than all other sharp-tipped polymer pens. These supports partially or fully offset stamp weights thereby also serving as a leveling system. We investigated a series of v-shaped polymer pens with known height differences to control relative vertical positions of each polymer pen precisely at the sub-20 nm scale mimicking a high-precision scanning stage. In doing so, we obtained linear-array patterns of alkanethiols with sub-50 nm to sub-500 nm line widths and minimum sub-20 nm line width tunable increments. The CLL pattern line widths were in agreement with those predicted by simulations. Our results suggest that through informed design of a stamp support system and tuning of polymer-pen base widths, throughput can be increased by eliminating the need for a scanning stage system in PPCLL without sacrificing precision. To demonstrate functional microarrays patterned by PPCLL, we inserted probe DNA into PPCLL patterns and observed hybridization by complementary target sequences.

Tumor-Targeted Multimodal Optical Imaging with Versatile Cadmium-Free Quantum Dots.

The rapid development of fluorescence imaging technologies requires concurrent improvements in the performance of fluorescent probes. Quantum dots have been extensively used as an imaging probe in various research areas because of their inherent advantages based on unique optical and electronic properties. However, their clinical translation has been limited by the potential toxicity especially from cadmium. Here, a versatile bioimaging probe is developed by using highly luminescent cadmium-free CuInSe2/ZnS core/shell quantum dots conjugated with CGKRK (Cys-Gly-Lys-Arg-Lys) tumor-targeting peptides. This probe exhibits excellent photostability, reasonably long circulation time, minimal toxicity, and strong tumor-specific homing property. The most important feature of this probe is that it shows distinctive versatility in tumor-targeted multimodal imaging including near-infrared, time-gated, and two-photon imaging in different tumor models. In a glioblastoma mouse model, the targeted probe clearly denotes tumor boundaries and positively labels a population of diffusely infiltrating tumor cells, suggesting its utility in precise tumor detection during surgery. This work lays a foundation for potential clinical translation of the probe.

Small GSH-Capped CuInS2 Quantum Dots: MPA-Assisted Aqueous Phase Transfer and Bioimaging Applications.

An efficient ligand exchange strategy for aqueous phase transfer of hydrophobic CuInS2/ZnS quantum dots was developed by employing glutathione (GSH) and mercaptopropionic acid (MPA) as the ligands. The whole process takes less than 20 min and can be scaled up to gram amount. The material characterizations show that the final aqueous soluble samples are solely capped with GSH on the surface. Importantly, these GSH-capped CuInS2/ZnS quantum dots have small size (hydrodynamic diameter <10 nm), moderate fluorescent properties (up to 34%) as well as high stability in aqueous solutions (stable for more than three months in 4 °C without any significant fluorescence quenching). Moreover, this ligand exchange strategy is also versatile for the aqueous phase transfer of other hydrophobic quantum dots, for instance, CuInSe2 and CdSe/ZnS quantum dots. We further demonstrated that GSH-capped quantum dots could be suitable fluorescence markers to penetrate cell membrane and image the cells. In addition, the GSH-capped CuInS2 quantum dots also have potential use in other fields such as photocatalysis and quantum dots sensitized solar cells.