Topographical Patterning Research Articles

Microstructure versus topography: the impact of crystallographic substrate modification during ultrashort pulsed direct laser interference patterning on the antibacterial properties of Cu

Introduction: Topographic surface patterning in the micro- and nanometer scale has evolved into a well applied approach in surface functionalization following biomimetic blueprints from nature. Depending on the production process an additional impact of process-related substrate modification has to be considered in functional surface optimization. This is especially true in case of antimicrobial applications of Cu surfaces where a modification of the substrate properties might impact bactericidal efficiency.Methods: In this regard, the effect of ultrashort pulsed direct laser interference patterning on the microstructure of pure Cu and resulting antimicrobial properties was investigated alongside line-like patterning in the scale of single bacterial cells.Results and Discussion: The process-induced microstructure modification was shown to play an important role in corrosion processes on Cu surfaces in saline environment, whereas the superficial microstructure impacts both corrosive interaction and ion emission. Surprisingly, antimicrobial efficiency is not predominantly following deviating trends in Cu ion release rates but rather depends on surface topography and wettability, which was shown to be impacted by the substrate microstructure state, as well. This highlights the need of an in-depth understanding on how different surface properties are simultaneously modulated during laser processing and how their interaction has to be designed to acquire an effective surface optimization e.g., to agitate active antimicrobial surface functionalization.

Contact angle hysteresis due to electric inhomogeneity of topographical patterning of dielectric layer in electrowetting

Despite the crucial role of contact angle hysteresis (CAH) in various industrial processes, there is a lack of approaches to the dynamic modulation of CAH. Herein, dielectric layers in electrowetting systems are fabricated with long-range and low-magnitude topographical patterning of the dielectric layer to generate an inhomogeneous electric field at the solid-liquid interface. Reversible and dynamic modulation of CAH is implemented while avoiding possible liquid pinning. The outcome of this work enables the programming of CAH and expands its application in the modulation of solid-liquid interactions.

Topographical surface patterning with block copolymer stamps

We explore the potential of stamps of the block copolymer (BCP) polystyrene-block-poly (2-vinylpyridine) (PS-b-P2VP) for the parallel lithographic generation of topographic surface patterns taking advantage of the specific BCP properties. A first example includes additive surface manufacturing by decal transfer microcontact lithography with PS-b-P2VP breath figure (BF) arrays. Suppression of entanglements at the internal BCP domain interfaces and the BF array morphology facilitate rupture of the thinnest wall segments in the BF arrays. The uppermost PS-b-P2VP BF array layers with heights of nearly 1 µm thus remained attached to counterpart substrates. Treatment with solvents selective to PS or P2VP enabled wettability switching of thus-functionalized surfaces. A second example includes subtractive surface manufacturing. We deposited holey films or stripe patterns of the perfluorinated alkylphosphonic acid F21-DDPA on glass substrates using PS-b-P2VP stamps penetrated by spongy mesopore systems generated by swelling-induced pore formation. The use of spongy-mesoporous stamps allows carrying out succesive stamping steps without ink replenishment. Subsequently, we topographically patterned the glass substrates by etching the parts of the glass substrates not protected by F21-DDPA with hydrofluoric acid (HF) solution. Within certain limitations, shape, area and depth of the topographic patterns may be adjusted by the etching durations. Circular PS-b-P2VP films were selectively deposited into hexagonal microwells and subjected to swelling-induced pore formation. Moreover, F21-DDPA-patterned glass substrates can be used to template the dewetting of polystyrene (PS). Arrays of PS droplets obtained in this way were in turn used as etch resists to produce arrays of glass nanoneedles. Hence, BCP stamps may be exploited to produce topographically patterned surfaces with different architectures for wettability management or graphoepitaxy.

A novel topographically patterned MXene@MnO2/PVDF piezo-active hybrid for flexible real-time and sensitive force sensor

Flexible force sensors capable of real-time and sensitive analysis have gained immense popularity because of their great potential to be integrated into potable and wearable electronics. Herein, a novel flexible force sensor constructing from topographically patterned piezo-active MXene@MnO2/poly(vinylidene fluoride) (PVDF) hybrid that allows for real-time precise pressure detection is proposed. Benefiting from the synergistic effect of topographical patterning and nano-reinforcement filling, the piezo-active hybrids with convex microarrays patterns demonstrate outstanding piezoelectric coefficient d33 (49 pC/N) and output upon mechanical stimuli, enabling the resultant force sensors with high voltage sensitivity of 0.450 V/N, fast response time of 16–19 ms and excellent operation stability. Furthermore, the feasibility of sensors in effectively detecting water droplets, gas flow and finger bending is demonstrated, confirming their viability for real applications.

Gradients of striatal function in antipsychotic-free first-episode psychosis and schizotypy

Both psychotic illness and subclinical psychosis-like experiences (PLEs) have been associated with cortico-striatal dysfunction. This work has largely relied on a discrete parcellation of the striatum into distinct functional areas, but recent evidence suggests that the striatum comprises multiple overlapping and smoothly varying gradients (i.e., modes) of functional organization. Here, we investigated two of these functional connectivity modes, previously associated with variations in the topographic patterning of cortico-striatal connectivity (first-order gradient), and dopaminergic innervation of the striatum (second-order gradient), and assessed continuities in striatal function from subclinical to clinical domains. We applied connectopic mapping to resting-state fMRI data to obtain the first-order and second-order striatal connectivity modes in two distinct samples: (1) 56 antipsychotic-free patients (26 females) with first-episode psychosis (FEP) and 27 healthy controls (17 females); and (2) a community-based cohort of 377 healthy individuals (213 females) comprehensively assessed for subclinical PLEs and schizotypy. The first-order “cortico-striatal” and second-order “dopaminergic” connectivity gradients were significantly different in FEP patients compared to controls bilaterally. In the independent sample of healthy individuals, variations in the left first-order “cortico-striatal” connectivity gradient were associated with inter-individual differences in a factor capturing general schizotypy and PLE severity. The presumed cortico-striatal connectivity gradient was implicated in both subclinical and clinical cohorts, suggesting that variations in its organization may represent a neurobiological trait marker across the psychosis continuum. Disruption of the presumed dopaminergic gradient was only noticeable in patients, suggesting that neurotransmitter dysfunction may be more apparent to clinical illness.

Biomimetic Substrate to Probe Dynamic Interplay of Topography and Stiffness on Cardiac Fibroblast Activation.

Materials with the ability to change properties can expand the capabilities of in vitro models of biological processes and diseases as it has become increasingly clear that static, stiff materials with smooth surfaces fall short in recapitulating the in vivo cellular microenvironment. Here, we introduce a patterned material that can be rapidly stiffened and softened in situ in response to an external magnetic field through the addition of magnetic inclusions into a soft silicone elastomer with topographic surface patterning. This substrate can be used for cell culture to investigate short-term cellular responses to dynamic stiffening or softening and the interaction with topography that encourages cells to assume a specific morphology. We investigated short-term cellular responses to dynamic stiffening or softening in human ventricular cardiac fibroblasts. Our results indicate that the combination of dynamic changes in stiffness with and without topographic cues induces different effects on the alignment and activation or deactivation of myofibroblasts. Cells cultured on patterned substrates exhibited a more aligned morphology than cells cultured on flat material; moreover, cell alignment was not dependent on substrate stiffness. On a patterned substrate, there was no significant change in the number of activated myofibroblasts when the material was temporally stiffened, but temporal softening caused a significant decrease in myofibroblast activation (50% to 38%), indicating a competing interaction of these characteristics on cell behavior. This material provides a unique in vitro platform to observe the time-dependent dynamics of cells by better mimicking more complex behaviors and realistic microenvironments for investigating biological processes, such as the development of fibrosis.

Submicron Topographically Patterned 3D Substrates Enhance Directional Axon Outgrowth of Dorsal Root Ganglia Cultured Ex Vivo.

A peripheral nerve injury results in disruption of the fiber that usually protects axons from the surrounding environment. Severed axons from the proximal nerve stump are capable of regenerating, but axons are exposed to a completely new environment. Regeneration recruits cells that produce and deposit key molecules, including growth factor proteins and fibrils in the extracellular matrix (ECM), thus changing the chemical and geometrical environment. The regenerating axons thus surf on a newly remodeled micro-landscape. Strategies to enhance and control axonal regeneration and growth after injury often involve mimicking the extrinsic cues that are found in the natural nerve environment. Indeed, nano- and micropatterned substrates have been generated as tools to guide axons along a defined path. The mechanical cues of the substrate are used as guides to orient growth or change the direction of growth in response to impediments or cell surface topography. However, exactly how axons respond to biophysical information and the dynamics of axonal movement are still poorly understood. Here we use anisotropic, groove-patterned substrate topography to direct and enhance sensory axonal growth of whole mouse dorsal root ganglia (DRG) transplanted ex vivo. Our results show significantly enhanced and directed growth of the DRG sensory fibers on the hemi-3D topographic substrates compared to a 0 nm pitch, flat control surface. By assessing the dynamics of axonal movement in time-lapse microscopy, we found that the enhancement was not due to increases in the speed of axonal growth, but to the efficiency of growth direction, ensuring axons minimize movement in undesired directions. Finally, the directionality of growth was reproduced on topographic patterns fabricated as fully 3D substrates, potentially opening new translational avenues of development incorporating these specific topographic feature sizes in implantable conduits in vivo.

Hierarchically Fabricated Amyloid Fibers via Evaporation-Induced Self-Assembly.

Multiscale hierarchical nano- and microstructures of amyloid fibrils are fabricated by evaporation-induced self-assembly combined with topographic surface patterning techniques. The continuous stick-and-slip motion induces uniaxial alignment of amyloid fibrils characterized by high orientational order during the drying process. The optical textures of the resultant amyloid aggregates are directly observed by polarized optical microscopy (POM) and atomic force microscopy (AFM). The resulting fiber structure can be tuned by varying the width of the topographic pattern, e.g., the microchannel width, inducing different separation between the deposited amyloid fibers on the glass substrate. Additionally, amyloid fibrils are decorated with gold nanoparticles to produce conductive microwires showing good conductivity (∼10-3 S/m). The finely controlled deposited amyloid fibers presented here can show a way to use naturally-abundant biomaterials for practical applications such as nanowires and sensors.

Spatial Control of Condensation: The Past, the Present, and the Future

AbstractSpatial control of condensation/nucleation is important for applications such as capillary force‐driven parallelized device alignment, suppression of chain‐reaction ice‐formation, and energy generation in steam cycles or dew collection to mitigate water scarcity. However, the nucleation is inherently random, limiting the possibilities to achieve defect‐free and durable spatial control. The two well‐known strategies for spatial control are chemical and topographical patterning, which can both promote nucleation in predetermined regions by spatially shifting the probability distribution for nucleation. Historical developments of the field and studies that focus on or utilize spatial control of condensation are presented herein. The studies are divided according to the strategy and length scale of surface patterning, from nanometer to millimeter. In addition to the two well‐known strategies, supersaturation is identified as a third strategy. Their advantages are discussed together with future directions of spatial control with a focus on stimuli‐responsive spatial control and the importance of transitioning to control of liquids other than water.

Parahippocampal latrophilin-2 (ADGRL2) expression controls topographical presubiculum to entorhinal cortex circuit connectivity.

Brain circuits are comprised of distinct interconnected neurons that are assembled by synaptic recognition molecules presented by defined pre- and post-synaptic neurons. This cell-cell recognition process is mediated by varying cellular adhesion molecules, including the latrophilin family of adhesion G-protein-coupled receptors. Focusing on parahippocampal circuitry, we find that latrophilin-2 (Lphn2; gene symbol ADGRL2) is specifically enriched in interconnected subregions of the medial entorhinal cortex (MEC), presubiculum (PrS), and parasubiculum (PaS). Retrograde viral tracing from the Lphn2-enriched region of the MEC reveals unique topographical patterning of inputs arising from the PrS and PaS that mirrors Lphn2 expression. Using a Lphn2 conditional knockout mouse model, we find that deletion of MEC Lphn2 expression selectively impairs retrograde viral labeling of inputs arising from the ipsilateral PrS. Combined with analysis of Lphn2 expression within the MEC, this study reveals Lphn2 to be selectively expressed by defined cell types and essential for MEC-PrS circuit connectivity.

Micro and Nanopatterned Ion Exchange Membranes for Applications in Electrochemical Devices

Topographical patterns on ion exchange membranes’ (IEMs’) surfaces have been show to improve the performance and energy efficiency in electrochemical energy conversion and water treatment processes. Substituting flat IEMs with patterned/profiled IEMs in electrodialysis reduces spacer channel mass transfer limitations by increasing the interfacial area between the liquid solution and membrane materials. Additionally, the extended surface of the profiled cation-exchange and anion-exchange membranes augment the spacer channel conductivity without obfuscating bulk liquid flow. Topographically patterned ion exchange membranes are fabricated using several methodologies that include photo-/soft-lithography, hot pressing, solution casting and 3D printing. The minimum lateral feature size that can be obtained by these methods is practically on the order of a few microns and potentially could be as low as few hundred nanometers. Block copolymer (BCP) lithography is a nanopatterning platform that generates lateral feature sizes of 5 to 50 nm. Thin films of diblock copolymers can self-assemble on a neutral layer resulting in lamella, cylinder, or sphere morphologies when the film is thermal or solvent vapor annealed. The morphology of the film primarily depends on volume fraction (fA, fB) of each block and interaction parameter between the blocks (χ). Poly(styrene-block-methylmethacrylate) (PS-b-PMMA) is one of the most studied block copolymer for BCP lithography because it form perpendicular aligned nanostructures via thermal annealing without topcoat layers to control the free surface interfacial energy. The etch contrast between the styrene and MMA blocks also makes it easier to transfer the pattern onto substrates – especially if sequential infiltration synthesis (SIS) is used.In this work, for the first time, a method to prepare nanopatterned IEMs using BCP lithography is reported. The SIS process post PS-b-PMMA self-assembly converts MMA into alumina creating organic-inorganic hard mask. The styrene block is removed via oxygen plasma in a reactive ion-etcher leaving behind a nanoporous alumina template on a silicon wafer. Sulfonated poly(arylene ether ether ketone) (SPEEK) is dropcasted onto the nanoporous alumina and the solvent is evaporated. Once releasing the SPEEK from the substrate, a nanopatterned cation-exchange membrane is attained. The electrochemical properties of the nanopatterned membrane such as conductivity, permselectivity and transference number are measured and compared to a flat, non-patterned membrane. The efficiency of these nanopatterned membranes for simple electrochemical devices, like a hydrogen pump, will also be presented.This work is supported from NSF Award # 1703307. Figure 1

Guided accumulation of active particles by topological design of a second-order skin effect

Collective guidance of out-of-equilibrium systems without using external fields is a challenge of paramount importance in active matter, ranging from bacterial colonies to swarms of self-propelled particles. Designing strategies to guide active matter and exploiting enhanced diffusion associated to its motion will provide insights for application from sensing, drug delivery to water remediation. However, achieving directed motion without breaking detailed balance, for example by asymmetric topographical patterning, is challenging. Here we engineer a two-dimensional periodic topographical design with detailed balance in its unit cell where we observe spontaneous particle edge guidance and corner accumulation of self-propelled particles. This emergent behaviour is guaranteed by a second-order non-Hermitian skin effect, a topologically robust non-equilibrium phenomenon, that we use to dynamically break detailed balance. Our stochastic circuit model predicts, without fitting parameters, how guidance and accumulation can be controlled and enhanced by design: a device guides particles more efficiently if the topological invariant characterizing it is non-zero. Our work establishes a fruitful bridge between active and topological matter, and our design principles offer a blueprint to design devices that display spontaneous, robust and predictable guided motion and accumulation, guaranteed by out-of-equilibrium topology.

Interaction of micropatterned topographical and biochemical cues to direct neurite growth from spiral ganglion neurons

Functional outcomes with neural prosthetic devices, such as cochlear implants, are limited in part due to physical separation between the stimulating elements and the neurons they stimulate. One strategy to close this gap aims to precisely guide neurite regeneration to position the neurites in closer proximity to electrode arrays. Here, we explore the ability of micropatterned biochemical and topographic guidance cues, singly and in combination, to direct the growth of spiral ganglion neuron (SGN) neurites, the neurons targeted by cochlear implants. Photopolymerization of methacrylate monomers was used to form unidirectional topographical features of ridges and grooves in addition to multidirectional patterns with 90o angle turns. Microcontact printing was also used to create similar uni- and multi-directional patterns of peptides on polymer surfaces. Biochemical cues included peptides that facilitate (laminin, LN) or repel (EphA4-Fc) neurite growth. On flat surfaces, SGN neurites preferentially grew on LN-coated stripes and avoided EphA4-Fc-coated stripes. LN or EphA4-Fc was selectively adsorbed onto the ridges or grooves to test the neurite response to a combination of topographical and biochemical cues. Coating the ridges with EphA4-Fc and grooves with LN lead to enhanced SGN alignment to topographical patterns. Conversely, EphA4-Fc coating on the grooves or LN coating on the ridges tended to disrupt alignment to topographical patterns. SGN neurites respond to combinations of topographical and biochemical cues and surface patterning that leverages both cues enhance guided neurite growth.

User-friendly chemical patterning with digital light projection polymer brush photolithography

Patterned polymeric coatings are broadly relevant for all areas of bioengineering: anti-biofouling, controlled protein adsorption, guided cell growth, and many more. This contribution describes a robust topographical and chemical patterning platform that combines an LED digital light projector with oxygen-tolerant light-mediated polymerization to design advanced surfaces on the micron scale and in mild ambient conditions. The user-friendly nature of this approach is targeted towards bringing complex chemical patterning abilities based on surface-tethered polymers into the hands of non-experts and enabling both fundamental and applied studies related to patterned surfaces in bioengineering.

Enhanced Biocompatibility and Differentiation Capacity of Mesenchymal Stem Cells on Poly(dimethylsiloxane) by Topographically Patterned Dopamine.

Controlling the behavior of mesenchymal stem cells (MSCs) through topographic patterns is an effective approach for stem cell studies. We, herein, reported a facile method to create a dopamine (DA) pattern on poly(dimethylsiloxane) (PDMS). The topography of micropatterned DA was produced on PDMS after plasma treatment. The grid-topographic-patterned surface of PDMS-DA (PDMS-DA-P) was measured for adhesion force and Young's modulus by atomic force microscopy. The surface of PDMS-DA-P demonstrated less stiff and more elastic characteristics compared to either nonpatterned PDMS-DA or PDMS. The PDMS-DA-P evidently enhanced the differentiation of MSCs into various tissue cells, including nerve, vessel, bone, and fat. We further designed comprehensive experiments to investigate adhesion, proliferation, and differentiation of MSCs in response to PDMS-DA-P and showed that the DA-patterned surface had good biocompatibility and did not activate macrophages or platelets in vitro and had low foreign body reaction in vivo. Besides, it protected MSCs from apoptosis as well as excessive reactive oxygen species (ROS) generation. Particularly, the patterned surface enhanced the differentiation capacity of MSCs toward neural and endothelial cells. The stromal cell-derived factor-1α/CXantiCR4 pathway may be involved in mediating the self-recruitment and promoting the differentiation of MSCs. These findings support the potential application of PDMS-DA-P in either cell treatment or tissue repair.

Novel Ultra Localized and Dense Nitrogen Delta-Doping in Diamond for Advanced Quantum Sensing.

We introduce and demonstrate a new approach for nitrogen-vacancy (NV) patterning in diamond, achieving a deterministic, nanometer-thin, and dense delta-doped layer of negatively charged NV centers in diamond. We employed a pure nitridation stage using microwave plasma and a subsequent in situ diamond overgrowth. We present the highest reported nitrogen concentration in a delta-doped layer (1.8 × 1020 cm-3) while maintaining the pristine diamond crystal quality. This result combined with the large optically detected magnetic resonance contrast can pave the way toward highly sensitive NV-based magnetometers. We further employed this delta-doping technique on high-quality fabricated diamond nanostructures for realizing a topographic NV patterning in order to enhance the sensing and hyperpolarization capabilities of NV-based devices.

Influence of micropatterned substrates on keratocyte phenotype

Substrate topographic patterning is a powerful tool that can be used to manipulate cell shape and orientation. To gain a better understanding of the relationship between surface topography and keratocyte behavior, surface patterns consisting of linear aligned or orthogonally aligned microchannels were used. Photolithography and polymer molding techniques were used to fabricate micropatterns on the surface of polydimethylsiloxane (PDMS). Cells on linear aligned substrates were elongated and aligned in the channel direction, while cells on orthogonal substrates had a more spread morphology. Both linear and orthogonal topographies induced chromatin condensation and resulted in higher expressions of keratocyte specific genes and sulfated glycosaminoglycans (sGAG), compared with non-patterned substrates. However, despite differences in cell morphology and focal adhesions, many genes associated with a native keratocyte phenotype, such as keratocan and ALDH3A1, remain unchanged on the different patterned substrates. This information could be used to optimize substrates for keratocyte culture and to develop scaffolds for corneal regeneration.

Vascular cell responses to silicone surfaces grafted with heparin-like polymers: surface chemical composition vs. topographic patterning.

Heparin-like polymers are promising synthetic materials with biological functionalities, such as anticoagulant ability, growth factor binding to regulate cellular functions, and inflammation mediation, similar to heparin. The biocompatibility of heparin-like polymers with well-defined chemical structures has inspired many researchers to design heparin-like surfaces to explore their biological applications. The concept of the recombination of functional heparin structural units (sulfonate- and glyco-containing units) was proven to be successful in designing heparin-mimicking surfaces. However, besides surface structural units, topographic patterning is also an important contributor to the biological activity of the surfaces modified with heparin-like polymers. In this work, both surface structural units and topographic patterning were taken into account to investigate the vascular cell behaviors on the silicone surfaces. A facile method for the production of patterned bromine-containing polydimethylsiloxane surface (PDMS-Br) was developed from a one-step multicomponent thermocuring procedure and replica molding using a nanohole-arrayed silicon template. Different structural units of heparin-like polymers, i.e. homopolymer of sulfonate-containing sodium 4-vinylbenzenesulfonate (pSS), homopolymer of glyco-containing 2-(methacrylamido)glucopyranose (pMAG), and copolymers of MAG and SS (pSG), were then introduced on the flat and patterned PDMS-Br surface using visible light-induced graft polymerization. For the flat surfaces, compared with the PDMS-Br surface, pSS-grafted and pSG-grafted surfaces significantly increased cell densities of both human umbilical vein endothelial cells (HUVECs) and human umbilical vein smooth muscle cells (HUVSMCs), indicating that they are "vascular cell-friendly". In contrast, the pMAG-grafted surface showed decreased cell attachment of both HUVECs and HUVSMCs, indicating that the pMAG-grafted surface is "vascular cell-resistant". Moreover, surface topographic patterning enhanced the cell responses to the corresponding flat surfaces. That is to say, surface patterning can make the "vascular cell-friendly" surface still friendly, and the "vascular cell-resistant" surface much more resistant. The combination of surface structural units and topographic patterning shows promise in the preparation of new heparin-like surfaces with improved cell compatibility that is suitable for blood-compatible biomaterials.

Compact pulsed thulium-doped fiber laser for topographical patterning of hydrogels

We report the generation of high energy 2 μm picosecond pulses from a thulium-doped fiber master oscillator power amplifier system. The all-fiber configuration was realized by a flexible large-mode area photonic crystal fiber (LMA-PCF). The amplifier output is a linearly-polarized 1.5 ns, 100 kHz pulse train with a pulse energy of up to 250 μJ. Pulse compression was achieved with (2+2)-pass chirped volume Bragg grating (CVBG) to obtain a 2.8 ps pulse width with a total pulse energy of 46 μJ. The overall system compactness was enabled by the all-fiber amplifier design and the multi-pass CVBG-based compressor. The laser output was then used to demonstrate high-speed direct-writing capability on a temperature-sensitive biomaterial to change its topography (i.e. fabricate microchannels, foams and pores). The topographical modifications of biomaterials are known to influence cell behavior and fate which is potentially useful in many cell and tissue engineering applications.

Sub-10 nm Resolution Patterning of Pockets for Enzyme Immobilization with Independent Density and Quasi-3D Topography Control.

The ability to precisely control the localization of enzymes on a surface is critical for several applications including biosensing, bionanoreactors, and single molecule studies. Despite recent advances, fabrication of enzyme patterns with resolution at the single enzyme level is limited by the lack of lithography methods that combine high resolution, compatibility with soft, polymeric structures, ease of fabrication, and high throughput. Here, a method to generate enzyme nanopatterns (using thermolysin as a model system) on a polymer surface is demonstrated using thermochemical scanning probe lithography (tc-SPL). Electrostatic immobilization of negatively charged sulfonated enzymes occurs selectively at positively charged amine nanopatterns produced by thermal deprotection of amines along the side-chain of a methacrylate-based copolymer film via tc-SPL. This process occurs simultaneously with local thermal quasi-3D topographical patterning of the same polymer, offering lateral sub-10 nm resolution, and vertical 1 nm resolution, as well as high throughput (5.2 × 104 μm2/h). The obtained single-enzyme resolution patterns are characterized by atomic force microscopy (AFM) and fluorescence microscopy. The enzyme density, the surface passivation, and the quasi-3D arbitrary geometry of these patterned pockets are directly controlled during the tc-SPL process in a single step without the need of markers or masks. Other unique features of this patterning approach include the combined single-enzyme resolution over mm2 areas and the possibility of fabricating enzymes nanogradients.