Correction: Direct laser writing-enabled 3D printing strategies for microfluidic applications.

Correction for 'Direct laser writing-enabled 3D printing strategies for microfluidic applications' by Olivia M. Young et al., Lab Chip, 2024, DOI: https://doi.org/10.1039/D3LC00743J.

Direct laser writing-enabled 3D printing strategies for microfluidic applications.

Over the past decade, additive manufacturing-or "three-dimensional (3D) printing"-has attracted increasing attention in the Lab on a Chip community as a pathway to achieve sophisticated system architectures that are difficult or infeasible to fabricate via conventional means. One particularly promising 3D manufacturing technology is "direct laser writing (DLW)", which leverages two-photon (or multi-photon) polymerization (2PP) phenomena to enable high geometric versatility, print speeds, and precision at length scales down to the 100 nm range. Although researchers have demonstrated the potential of using DLW for microfluidic applications ranging from organ on a chip and drug delivery to micro/nanoparticle processing and soft microrobotics, such scenarios present unique challenges for DLW. Specifically, microfluidic systems typically require macro-to-micro fluidic interfaces (e.g., inlet and outlet ports) to facilitate fluidic loading, control, and retrieval operations; however, DLW-based 3D printing relies on a micron-to-submicron-sized 2PP volume element (i.e., "voxel") that is poorly suited for manufacturing these larger-scale fluidic interfaces. In this Tutorial Review, we highlight and discuss the four most prominent strategies that researchers have developed to circumvent this trade-off and realize macro-to-micro interfaces for DLW-enabled microfluidic components and systems. In addition, we consider the possibility that-with the advent of next-generation commercial DLW printers equipped with new dynamic voxel tuning, print field, and laser power capabilities-the overall utility of DLW strategies for Lab on a Chip fields may soon expand dramatically.

GEOMETRIC DETERMINANTS OF CELL VIABILITY FOR 3D-PRINTED HOLLOW MICRONEEDLE ARRAY-MEDIATED DELIVERY.

A wide range of emerging biomedical applications and clinical interventions rely on the ability to deliver living cells via hollow, high-aspect-ratio microneedles. Recently, microneedle arrays (MNA) have gained increasing interest due to inherent benefits for drug delivery; however, studies exploring the potential to harness such advantages for cell delivery have been impeded due to the difficulties in manufacturing high-aspect-ratio MNAs suitable for delivering mammalian cells. To bypass these challenges, here we leverage and extend our previously reported hybrid additive manufacturing (or "three-dimensional (3D) printing) strategy-i.e., the combined the "Vat Photopolymerization (VPP)" technique, "Liquid Crystal Display (LCD)" 3D printing with "Two-Photon Direct Laser Writing (DLW)"-to 3D print hollow MNAs that are suitable for cell delivery investigations. Specifically, we 3D printed four sets of 650 µm-tall MNAs corresponding to needle-specific inner diameters (IDs) of 25 µm, 50 µm, 75 µm, and 100 µm, and then examined the effects of these MNAs on the post-delivery viability of both dendritic cells (DCs) and HEK293 cells. Experimental results revealed that the 25 µm-ID case led to a statistically significant reduction in post-MNA-delivery cell viability for both cell types; however, MNAs with needle-specific IDs ≥ 50 µm were statistically indistinguishable from one another as well as conventional 32G single needles, thereby providing an important benchmark for MNA-mediated cell delivery.

FABRICATION OF MULTILUMEN MICROFLUIDIC TUBING FOR EX SITU DIRECT LASER WRITING.

Among the numerous additive manufacturing or "three-dimensional (3D) printing" techniques, two-photon Direct Laser Writing (DLW) is distinctively suited for applications that demand high geometric versatility with micron-to-submicron-scale feature resolutions. Recently, "ex situ DLW (esDLW)" has emerged as a powerful approach for printing 3D microfluidic structures directly atop meso/macroscale fluidic tubing that can be manipulated by hand; however, difficulties in creating custom esDLW-compatible multilumen tubing at such scales has hindered progress. To address this impediment, here we introduce a novel methodology for fabricating submillimeter multilumen tubing for esDLW 3D printing. Preliminary fabrication results demonstrate the utility of the presented strategy for resolving 743 µm-in-diameter tubing with three lumens-each with an inner diameter (ID) of 80 µm. Experimental results not only revealed independent flow of discrete fluorescently labelled fluids through each of the three lumens, but also effective esDLW-printing of a demonstrative 3D "MEMS" microstructure atop the tubing. These results suggest that the presented approach could offer a promising pathway to enable geometrically sophisticated microfluidic systems to be 3D printed with input and/or output ports fully sealed to multiple, distinct lumens of fluidic tubing for emerging applications in fields ranging from drug delivery and medical diagnostics to soft surgical robotics.

TOWARD CONTROLLED-RELEASE DRUG DELIVERY MICROCARRIERS ENABLED BY DIRECT LASER WRITING 3D PRINTING.

Controlled-release, and especially long-acting, drug delivery systems hold promise for improving treatments for numerous medical conditions. Previously, we reported an additive manufacturing or "three-dimensional (3D) printing" approach for fabricating liquid-core-shell-cap microcarriers comprising standard photoresists. Here we explore the potential to extend this strategy to achieve microcarriers comprising biodegradable materials as a new pathway to controlled-release drug delivery options. Specifically, we investigate the use of "Two-Photon Direct Laser Writing (DLW)" as a means to 3D print microcarriers composed of: (i) a bottle-shaped "shell" with an orifice, (ii) an aqueous liquid "core", and (iii) a biodegradable "cap". The cap, which is DLW-printed directly onto the shell's orifice, is designed to degrade over time in the body-e.g., with degradation time proportional to cap thickness-to ultimately facilitate release of the liquid core at desired time points. Fabrication results based on the use of a biodegradable poly(ethylene glycol) diacrylate (PEGDA) photomaterial for the cap revealed that shell designs incorporating microfluidic obstruction structures appeared to limit undesired entry of the liquid-phase PEGDA into the shell (i.e., directly preceding cap printing), thereby resulting in improved retention of the liquid core after completion of the cap printing process. These results mark an important first step toward evaluating the utility of the presented DLW 3D printing strategy for possible drug delivery applications.

A 3D-MICROPRINTED COAXIAL NOZZLE FOR FABRICATING LONG, FLEXIBLE MICROFLUIDIC TUBING.

A variety of emerging applications, particularly those in medical and soft robotics fields, are predicated on the ability to fabricate long, flexible meso/microfluidic tubing with high customization. To address this need, here we present a hybrid additive manufacturing (or "three-dimensional (3D) printing") strategy that involves three key steps: (i) using the "Vat Photopolymerization (VPP) technique, "Liquid-Crystal Display (LCD)" 3D printing to print a bulk microfluidic device with three inlets and three concentric outlets; (ii) using "Two-Photon Direct Laser Writing (DLW)" to 3D microprint a coaxial nozzle directly atop the concentric outlets of the bulk microdevice, and then (iii) extruding paraffin oil and a liquid-phase photocurable resin through the coaxial nozzle and into a polydimethylsiloxane (PDMS) channel for UV exposure, ultimately producing the desired tubing. In addition to fabricating the resulting tubing-composed of polymerized photomaterial-at arbitrary lengths (e.g., > 10 cm), the distinct input pressures can be adjusted to tune the inner diameter (ID) and outer diameter (OD) of the fabricated tubing. For example, experimental results revealed that increasing the driving pressure of the liquid-phase photomaterial from 50 kPa to 100 kPa led to fluidic tubing with IDs and ODs of 291±99 µm and 546±76 µm up to 741±31 µm and 888±39 µm, respectively. Furthermore, preliminary results for DLW-printing a microfluidic "M" structure directly atop the tubing suggest that the tubing could be used for "ex situ DLW (esDLW)" fabrication, which would further enhance the utility of the tubing.

3D-Printed Microinjection Needle Arrays via a Hybrid DLP-Direct Laser Writing Strategy.

Microinjection protocols are ubiquitous throughout biomedical fields, with hollow microneedle arrays (MNAs) offering distinctive benefits in both research and clinical settings. Unfortunately, manufacturing-associated barriers remain a critical impediment to emerging applications that demand high-density arrays of hollow, high-aspect-ratio microneedles. To address such challenges, here, a hybrid additive manufacturing approach that combines digital light processing (DLP) 3D printing with "ex situ direct laser writing (esDLW)" is presented to enable new classes of MNAs for fluidic microinjections. Experimental results for esDLW-based 3D printing of arrays of high-aspect-ratio microneedles-with 30 µm inner diameters, 50 µm outer diameters, and 550 µm heights, and arrayed with 100 µm needle-to-needle spacing-directly onto DLP-printed capillaries reveal uncompromised fluidic integrity at the MNA-capillary interface during microfluidic cyclic burst-pressure testing for input pressures in excess of 250 kPa (n = 100 cycles). Ex vivo experiments perform using excised mouse brains reveal that the MNAs not only physically withstand penetration into and retraction from brain tissue but also yield effective and distributed microinjection of surrogate fluids and nanoparticle suspensions directly into the brains. In combination, the results suggest that the presented strategy for fabricating high-aspect-ratio, high-density, hollow MNAs could hold unique promise for biomedical microinjection applications.

Deterministic Lateral Displacement Using Hexagonally Arranged, Bottom-Up-Inspired Micropost Arrays.

Size-based separation of particles in microfluidic devices can be achieved using arrays of micro- or nanoscale posts using a technique known as deterministic lateral displacement (DLD). To date, DLD arrays have been limited to parallelogram or rotated-square arrangements of posts, with various post shapes having been explored in these two principal arrangements. This work examines a new DLD geometry based on patterning obtainable through self-assembly of single-layer nanospheres, which we call hexagonally arranged triangle (HAT) geometry. Finite element simulations are used to characterize the DLD separation properties of the HAT geometry. The relationship between the array angle, the gap spacing, and the critical diameter for separation is derived for the HAT geometry and expressed in a similar mathematical form as conventional parallelogram and rotated-square DLD arrays. At array angles <7°, HAT structures demonstrate smaller particle sorting capability (smaller critical diameter-to-gap spacing ratio) compared to published experimental results for parallelogram-type DLD arrays with circular posts. Experimental validation of DLD separation confirms the separation ability of the HAT array geometry. It is envisioned that this work will provide the first step toward future implementation of nanoscale DLD arrays fabricated by low-cost, bottom-up self-assembly approaches.

Bayesian Optimization for Design of Multi-Actuator Soft Catheter Robots.

Catheter-based diagnosis and therapy have grown increasingly in recent years due to their improved clinical outcomes including decreased morbidity, shorter recovery time and minimally invasiveness compared to open surgeries. Although the scalability, customizability, and diversity of soft catheter robots are widely recognized, designers and roboticists still lack comprehensive techniques for modeling and designing them. This difficulty arises due to their continuum nature, which makes characterizing the properties and predicting a soft catheter's behavior challenging, complicating robot design tasks. In this paper, we propose modeling multi-actuator soft catheters to enable alignment with desired vessel shapes near the target area. We develop mathematical models to simulate the catheter's positioning due to the moments exerted by multiple pneumatic actuators along the catheter and use those models to compare optimization approaches that can achieve catheter alignment along a desired vessel shape. Specifically, our approach proposes finding the optimal geometric and material properties for a multi-actuator soft catheter robot using a bi-level optimization framework. The upper-level optimization process uses a modified Bayesian technique to seek the optimal geometric and material properties of the soft catheter, which minimize the deviance of the actuated catheter from a desired vessel shape, while the lower-level optimization process uses a gradient-based technique to obtain the actuator moments required to achieve that vessel shape. The results demonstrate the capability of our proposed multi-actuator soft catheter to align with the desired vessel shapes, and show that the proposed framework which is in the context of Bayesian optimization has the potential to expedite the design process.

Fully 3D-printed soft robots with integrated fluidic circuitry.

The emergence of soft robots has presented new challenges associated with controlling the underlying fluidics of such systems. Here, we introduce a strategy for additively manufacturing unified soft robots comprising fully integrated fluidic circuitry in a single print run via PolyJet three-dimensional (3D) printing. We explore the efficacy of this approach for soft robots designed to leverage novel 3D fluidic circuit elements-e.g., fluidic diodes, "normally closed" transistors, and "normally open" transistors with geometrically tunable pressure-gain functionalities-to operate in response to fluidic analogs of conventional electronic signals, including constant-flow ["direct current (DC)"], "alternating current (AC)"-inspired, and preprogrammed aperiodic ("variable current") input conditions. By enabling fully integrated soft robotic entities (composed of soft actuators, fluidic circuitry, and body features) to be rapidly disseminated, modified on demand, and 3D-printed in a single run, the presented design and additive manufacturing strategy offers unique promise to catalyze new classes of soft robots.

3D printed vascular phantoms for high-resolution biophotonic image quality assessment via direct laser writing.

Fluorescence imaging techniques such as fluorescein angiography and fundus autofluorescence are often used to diagnose retinal pathologies; however, there are currently no standardized test methods for evaluating device performance. Here we present microstructured fluorescent phantoms fabricated using a submicron-scale three-dimensional printing technology, direct laser writing (DLW). We employ an in situ DLW technique to print 10 µm diameter microfluidic channels that support perfusions of fluorescent dyes. We then demonstrate how broadband photoresist fluorescence can be exploited to generate resolution targets and biomimetic models of retinal vasculature using standard DLW processes. The results indicate that these approaches show significant promise for generating better performance evaluation tools for fluorescence microscopy and imaging devices.

Physics-Informed Modeling and Control of Multi-Actuator Soft Catheter Robots.

Catheter-based endovascular interventional procedures have become increasingly popular in recent years as they are less invasive and patients spend less time in the hospital with less recovery time and less pain. These advantages have led to a significant growth in the number of procedures that are performed annually. However, it is still challenging to position a catheter in a target vessel branch within the highly complicated and delicate vascular structure. In fact, vessel tortuosity and angulation, which cause difficulties in catheterization and reaching the target site, have been reported as the main causes of failure in endovascular procedures. Maneuverability of a catheter for intravascular navigation is a key to reaching the target area; ability of a catheter to move within the target vessel during trajectory tracking thus affects to a great extent the length and success of the procedure. To address this issue, this paper models soft catheter robots with multiple actuators and provides a time-dependent model for characterizing the dynamics of multi-actuator soft catheter robots. Built on this model, an efficient and scalable optimization-based framework is developed for guiding the catheter to pass through arteries and reach the target where an aneurysm is located. The proposed framework models the deflection of the multi-actuator soft catheter robot and develops a control strategy for movement of catheter along a desired trajectory. This provides a simulation-based framework for selection of catheters prior to endovascular catheterization procedures, assuring that given a fixed design, the catheter is able to reach the target location. The results demonstrate the benefits that can be achieved by design and control of catheters with multiple number of actuators for navigation into small vessels.

3D microfluidics via cyclic olefin polymer-based in situ direct laser writing.

In situ direct laser writing (isDLW) strategies that facilitate the printing of three-dimensional (3D) nanostructured components directly inside of, and fully sealed to, enclosed microchannels are uniquely suited for manufacturing geometrically complex microfluidic technologies. Recent efforts have demonstrated the benefits of using micromolding and bonding protocols for isDLW; however, the reliance on polydimethylsiloxane (PDMS) leads to limited fluidic sealing (e.g., operational pressures <50-75 kPa) and poor compatibility with standard organic solvent-based developers. To bypass these issues, here we explore the use of cyclic olefin polymer (COP) as an enabling microchannel material for isDLW by investigating three fundamental classes of microfluidic systems corresponding to increasing degrees of sophistication: (i) "2.5D" functionally static fluidic barriers (10-100 µm in height), which supported uncompromised structure-to-channel sealing under applied input pressures of up to 500 kPa; (ii) 3D static interwoven microvessel-inspired structures (inner diameters < 10 µm) that exhibited effective isolation of distinct fluorescently labelled microfluidic flow streams; and (iii) 3D dynamically actuated microfluidic transistors, which comprised bellowed sealing elements (wall thickness = 500 nm) that could be actively deformed via an applied gate pressure to fully obstruct source-to-drain fluid flow. In combination, these results suggest that COP-based isDLW offers a promising pathway to wide-ranging fluidic applications that demand significant architectural versatility at submicron scales with invariable sealing integrity, such as for biomimetic organ-on-a-chip systems and integrated microfluidic circuits.

A facile multi-material direct laser writing strategy.

Direct laser writing (DLW) is a three-dimensional (3D) manufacturing technology that offers vast architectural control at submicron scales, yet remains limited in cases that demand microstructures comprising more than one material. Here we present an accessible microfluidic multi-material DLW (µFMM-DLW) strategy that enables 3D nanostructured components to be printed with average material registration accuracies of 100 ± 70 nm (ΔX) and 190 ± 170 nm (ΔY) - a significant improvement versus conventional multi-material DLW methods. Results for printing 3D microstructures with up to five materials suggest that µFMM-DLW can be utilized in applications that demand geometrically complex, multi-material microsystems, such as for photonics, meta-materials, and 3D cell biology.

3-D printed photoreceptor phantoms for evaluating lateral resolution of adaptive optics imaging systems.

With adaptive optics (AO), optical coherence tomography and scanning laser ophthalmoscopy imaging systems can resolve individual photoreceptor cells in living eyes, due to enhanced lateral spatial resolution. However, no standard test method exists for experimentally quantifying this parameter in ophthalmic AO imagers. Here, we present three-dimensional (3-D) printed phantoms, which enable the measurement of lateral resolution in an anatomically relevant manner. We used two-photon polymerization to fabricate two phantoms, which mimic the mosaic of cone photoreceptor outer segments at multiple retinal eccentricities. With these phantoms, we demonstrated that the resolution of two multimodal AO systems is similar to theoretical predictions, with some intriguing speckle effects.

Geometric Determinants of In-Situ Direct Laser Writing.

Direct laser writing (DLW) is a three-dimensional (3D) manufacturing technology that offers significant geometric versatility at submicron length scales. Although these characteristics hold promise for fields including organ modeling and microfluidic processing, difficulties associated with facilitating the macro-to-micro interfaces required for fluid delivery have limited the utility of DLW for such applications. To overcome this issue, here we report an in-situ DLW (isDLW) strategy for creating 3D nanostructured features directly inside of-and notably, fully sealed to-sol-gel-coated elastomeric microchannels. In particular, we investigate the role of microchannel geometry (e.g., cross-sectional shape and size) in the sealing performance of isDLW-printed structures. Experiments revealed that increasing the outward tapering of microchannel sidewalls improved fluidic sealing integrity for channel heights ranging from 10 µm to 100 µm, which suggests that conventional microchannel fabrication approaches are poorly suited for isDLW. As a demonstrative example, we employed isDLW to 3D print a microfluidic helical coil spring diode and observed improved flow rectification performance at higher pressures-an indication of effective structure-to-channel sealing. We envision that the ability to readily integrate 3D nanostructured fluidic motifs with the entire luminal surface of elastomeric channels will open new avenues for emerging applications in areas such as soft microrobotics and biofluidic microsystems.

A Role for 3D Printing in Kidney-on-a-Chip Platforms.

The advancement of "kidney-on-a-chip" platforms - submillimeter-scale fluidic systems designed to recapitulate renal functions in vitro - directly impacts a wide range of biomedical fields, including drug screening, cell and tissue engineering, toxicity testing, and disease modelling. To fabricate kidney-on-a-chip technologies, researchers have primarily adapted traditional micromachining techniques that are rooted in the integrated circuit industry; hence the term, "chip." A significant challenge, however, is that such methods are inherently monolithic, which limits one's ability to accurately recreate the geometric and architectural complexity of the kidney in vivo. Better reproduction of the anatomical complexity of the kidney will allow for more instructive modelling of physiological and pathophysiological events. Emerging additive manufacturing or "three-dimensional (3D) printing" techniques could provide a promising alternative to conventional methodologies. In this article, we discuss recent progress in the development of both kidney-on-a-chip platforms and state-of-the-art submillimeter-scale 3D printing methods, with a focus on biophysical and architectural capabilities. Lastly, we examine the potential for 3D printing-based approaches to extend the efficacy of kidney-on-a-chip systems.

Finger-powered microfluidic systems using multilayer soft lithography and injection molding processes.

Point-of-care (POC) and disposable biomedical applications demand low-power microfluidic systems with pumping components that provide controlled pressure sources. Unfortunately, external pumps have hindered the implementation of such microfluidic systems due to limitations associated with portability and power requirements. Here, we propose and demonstrate a 'finger-powered' integrated pumping system as a modular element to provide pressure head for a variety of advanced microfluidic applications, including finger-powered on-chip microdroplet generation. By utilizing a human finger for the actuation force, electrical power sources that are typically needed to generate pressure head were obviated. Passive fluidic diodes were designed and implemented to enable distinct fluids from multiple inlet ports to be pumped using a single actuation source. Both multilayer soft lithography and injection molding processes were investigated for device fabrication and performance. Experimental results revealed that the pressure head generated from a human finger could be tuned based on the geometric characteristics of the pumping system, with a maximum observed pressure of 7.6 ± 0.1 kPa. In addition to the delivery of multiple, distinct fluids into microfluidic channels, we also employed the finger-powered pumping system to achieve the rapid formation of both water-in-oil droplets (106.9 ± 4.3 µm in diameter) and oil-in-water droplets (75.3 ± 12.6 µm in diameter) as well as the encapsulation of endothelial cells in droplets without using any external or electrical controllers.

Microfluidic bead-based diodes with targeted circular microchannels for low Reynolds number applications.

Self-regulating fluidic components are critical to the advancement of microfluidic processors for chemical and biological applications, such as sample preparation on chip, point-of-care molecular diagnostics, and implantable drug delivery devices. Although researchers have developed a wide range of components to enable flow rectification in fluidic systems, engineering microfluidic diodes that function at the low Reynolds number (Re) flows and smaller scales of emerging micro/nanofluidic platforms has remained a considerable challenge. Recently, researchers have demonstrated microfluidic diodes that utilize high numbers of suspended microbeads as dynamic resistive elements; however, using spherical particles to block fluid flow through rectangular microchannels is inherently limited. To overcome this issue, here we present a single-layer microfluidic bead-based diode (18 µm in height) that uses a targeted circular-shaped microchannel for the docking of a single microbead (15 µm in diameter) to rectify fluid flow under low Re conditions. Three-dimensional simulations and experimental results revealed that adjusting the docking channel geometry and size to better match the suspended microbead greatly increased the diodicity (Di) performance. Arraying multiple bead-based diodes in parallel was found to adversely affect system efficacy, while arraying multiple diodes in series was observed to enhance device performance. In particular, systems consisting of four microfluidic bead-based diodes with targeted circular-shaped docking channels in series revealed average Di's ranging from 2.72 ± 0.41 to 10.21 ± 1.53 corresponding to Re varying from 0.1 to 0.6.

Dual-mode hydrodynamic railing and arraying of microparticles for multi-stage signal detection in continuous flow biochemical microprocessors.

Continuous flow particulate-based microfluidic processors are in critical demand for emerging applications in chemistry and biology, such as point-of-care molecular diagnostics. Challenges remain, however, for accomplishing biochemical assays in which microparticle immobilization is desired or required during intermediate stages of fluidic reaction processes. Here we present a dual-mode microfluidic reactor that functions autonomously under continuous flow conditions to: (i) execute multi-stage particulate-based fluidic mixing routines, and (ii) array select numbers of microparticles during each reaction stage (e.g., for optical detection). We employ this methodology to detect the inflammatory cytokine, interferon-gamma (IFN-γ), via a six-stage aptamer-based sandwich assay.