Alternative nano-lithographic tools for shell-isolated nanoparticle enhanced Raman spectroscopy substrates.

Chemically synthesized metal nanoparticles (MNPs) have been widely used as surface-enhanced Raman spectroscopy (SERS) substrates for monitoring catalytic reactions. In some applications, however, the SERS MNPs, besides being plasmonically active, can also be catalytically active and result in Raman signals from undesired side products. The MNPs are typically insulated with a thin (∼3 nm), in principle pin-hole-free shell to prevent this. This approach, which is known as shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS), offers many advantages, such as better thermal and chemical stability of the plasmonic nanoparticle. However, having both a high enhancement factor and ensuring that the shell is pin-hole-free is challenging because there is a trade-off between the two when considering the shell thickness. So far in the literature, shell insulation has been successfully applied only to chemically synthesized MNPs. In this work, we alternatively study different combinations of chemical synthesis (bottom-up) and lithographic (top-down) routes to obtain shell-isolated plasmonic nanostructures that offer chemical sensing capabilities. The three approaches we study in this work include (1) chemically synthesized MNPs + chemical shell, (2) lithographic substrate + chemical shell, and (3) lithographic substrate + atomic layer deposition (ALD) shell. We find that ALD allows us to fabricate controllable and reproducible pin-hole-free shells. We showcase the ability to fabricate lithographic SHINER substrates which report an enhancement factor of 7.5 × 103 ± 17% for our gold nanodot substrates coated with a 2.8 nm aluminium oxide shell. Lastly, by introducing a gold etchant solution to our fabricated SHINER substrate, we verified that the shells fabricated with ALD are truly pin-hole-free.

Fluorescent-Probe Characterization for Pore-Space Mapping with Single-Particle Tracking.

Porous solids often contain complex pore networks with pores of various sizes. Tracking individual fluorescent probes as they diffuse through porous materials can be used to characterize pore networks at tens of nanometers resolution. However, understanding the motion behavior of fluorescent probes in confinement is crucial to reliably derive pore network properties. Here, we introduce well-defined lithography-made model pores developed to study probe behavior in confinement. We investigated the influence of probe-host interactions on diffusion and trapping of confined single-emitter quantum-dot probes. Using the pH-responsiveness of the probes, we were able to largely suppress trapping at the pore walls. This enabled us to define experimental conditions for mapping of the accessible pore space of a one-dimensional pore array as well as a real-life polymerization-catalyst-support particle.

Low-Variance Surface-Enhanced Raman Spectroscopy Using Confined Gold Nanoparticles over Silicon Nanocones.

Surface-enhanced Raman spectroscopy (SERS) substrates are of utmost interest in the analyte detection of biological and chemical diagnostics. This is primarily due to the ability of SERS to sensitively measure analytes present in localized hot spots of the SERS nanostructures. In this work, we present the formation of 67 ± 6 nm diameter gold nanoparticles supported by vertically aligned shell-insulated silicon nanocones for ultralow variance SERS. The nanoparticles are obtained through discrete rotation glancing angle deposition of gold in an e-beam evaporating system. The morphology is assessed through focused ion beam tomography, energy-dispersive X-ray spectroscopy, and scanning electron microscopy. The optical properties are discussed and evaluated through reflectance measurements and finite-difference time-domain simulations. Lastly, the SERS activity is measured by benzenethiol functionalization and subsequent Raman spectroscopy in the surface scanning mode. We report a homogeneous analytical enhancement factor of 2.2 ± 0.1 × 107 (99% confidence interval for N = 400 grid spots) and made a comparison to other lithographically derived assemblies used in SERS. The strikingly low variance (4%) of our substrates facilitates its use for many potential SERS applications.

Droplet microreactor for high-throughput fluorescence-based measurements of single catalyst particle acidity.

The particles of heterogeneous catalysts differ greatly in size, morphology, and most importantly, in activity. Studying these catalyst particles in batch typically results in ensemble averages, without any information at the level of individual catalyst particles. To date, the study of individual catalyst particles has been rewarding but is still rather slow and often cumbersome1. Furthermore, these valuable in-depth studies at the single particle level lack statistical relevance. Here, we report the development of a droplet microreactor for high-throughput fluorescence-based measurements of the acidities of individual particles in fluid catalytic cracking (FCC) equilibrium catalysts (ECAT). This method combines systematic screening of single catalyst particles with statistical relevance. An oligomerization reaction of 4-methoxystyrene, catalyzed by the Brønsted acid sites inside the zeolite domains of the ECAT particles, was performed on-chip at 95 °C. The fluorescence signal generated by the reaction products inside the ECAT particles was detected near the outlet of the microreactor. The high-throughput acidity screening platform was capable of detecting ~1000 catalyst particles at a rate of 1 catalyst particle every 2.4 s. The number of detected catalyst particles was representative of the overall catalyst particle population with a confidence level of 95%. The measured fluorescence intensities showed a clear acidity distribution among the catalyst particles, with the majority (96.1%) showing acidity levels belonging to old, deactivated catalyst particles and a minority (3.9%) exhibiting high acidity levels. The latter are potentially of high interest, as they reveal interesting new physicochemical properties indicating why the particles were still highly acidic and reactive.

Nanoparticle Printing for Microfluidic Applications: Bipolar Electrochemistry and Localized Raman Sensing Spots.

The local integration of metal nanoparticle films on 3D-structured polydimethylsiloxane (PDMS)-based microfluidic devices is of high importance for applications including electronics, electrochemistry, electrocatalysis, and localized Raman sensing. Conventional processes to locally deposit and pattern metal nanoparticles require multiple steps and shadow masks, or access to cleanroom facilities, and therefore, are relatively imprecise, or time and cost-ineffective. As an alternative, we present an aerosol-based direct-write method, in which patterns of nanoparticles generated via spark ablation are locally printed with sub-mm size and precision inside of microfluidic structures without the use of lithography or other masking methods. As proof of principle, films of Pt or Ag nanoparticles were printed in the chambers of a multiplexed microfluidic device and successfully used for two different applications: Screening electrochemical activity in a high-throughput fashion, and localized sensing of chemicals via surface-enhanced Raman spectroscopy (SERS). The versatility of the approach will enable the generation of functional microfluidic devices for applications that include sensing, high-throughput screening platforms, and microreactors using catalytically driven chemical conversions.

Systematic characterization of cleanroom-free fabricated macrovalves, demonstrating pumps and mixers for automated fluid handling tuned for organ-on-chip applications.

Integrated valves enable automated control in microfluidic systems, as they can be applied for mixing, pumping and compartmentalization purposes. Such automation would be highly valuable for applications in organ-on-chip (OoC) systems. However, OoC systems typically have channel dimensions in the range of hundreds of micrometers, which is an order of magnitude larger than those of typical microfluidic valves. The most-used fabrication process for integrated, normally open polydimethylsiloxane (PDMS) valves requires a reflow photoresist that limits the achievable channel height. In addition, the low stroke volumes of these valves make it challenging to achieve flow rates of microliters per minute, which are typically required in OoC systems. Herein, we present a mechanical 'macrovalve' fabricated by multilayer soft lithography using micromilled direct molds. We demonstrate that these valves can close off rounded channels of up to 700 µm high and 1000 µm wide. Furthermore, we used these macrovalves to create a peristaltic pump with a pumping rate of up to 48 µL/min and a mixing and metering device that can achieve the complete mixing of a volume of 6.4 µL within only 17 s. An initial cell culture experiment demonstrated that a device with integrated macrovalves is biocompatible and allows the cell culture of endothelial cells over multiple days under continuous perfusion and automated medium refreshment.

Fluidic circuit board with modular sensor and valves enables stand-alone, tubeless microfluidic flow control in organs-on-chips.

Organs-on-chips are a unique class of microfluidic in vitro cell culture models, in which the in vivo tissue microenvironment is mimicked. Unfortunately, their widespread use is hampered by their operation complexity and incompatibility with end-user research settings. To address these issues, many commercial and non-commercial platforms have been developed for semi-automated culture of organs-on-chips. However, these organ-on-chip culture platforms each represent a closed ecosystem, with very little opportunity to interchange and integrate components from different platforms or to develop new ones. The translational organ-on-chip platform (TOP) is a multi-institutional effort to develop an open platform for automated organ-on-chip culture and integration of components from various developers. Central to TOP is the fluidic circuit board (FCB), a microfluidic plate with the form factor of a typical well plate. The FCB enables microfluidic control of multiple components like sensors or organ-on-chip devices through an interface based on openly available standards. Here, we report an FCB to integrate commercial and in-house developed components forming a stand-alone flow control system for organs-on-chips. The control system is able to achieve constant and pulsatile flow recirculation through a connected organ-on-chip device. We demonstrate that this system is able to automatically perfuse a heart-on-chip device containing co-cultures of cardiac tissues derived from human pluripotent stem cell-derived cardiomyocytes and monolayers of endothelial cells for five days. Altogether, we conclude that open technology platforms allow the integration of components from different sources to form functional and fit-for-purpose organ-on-chip systems. We anticipate that open platforms will play a central role in catalyzing and maturing further technological development of organ-on-chip culture systems.

Facilitating implementation of organs-on-chips by open platform technology.

Organ-on-chip (OoC) and multi-organs-on-chip (MOoC) systems have the potential to play an important role in drug discovery, disease modeling, and personalized medicine. However, most devices developed in academic labs remain at a proof-of-concept level and do not yet offer the ease-of-use, manufacturability, and throughput that are needed for widespread application. Commercially available OoC are easier to use but often lack the level of complexity of the latest devices in academia. Furthermore, researchers who want to combine different chips into MOoC systems are limited to one supplier, since commercial systems are not compatible with each other. Given these limitations, the implementation of standards in the design and operation of OoCs would strongly facilitate their acceptance by users. Importantly, the implementation of such standards must be carried out by many participants from both industry and academia to ensure a widespread acceptance and adoption. This means that standards must also leave room for proprietary technology development next to promoting interchangeability. An open platform with standardized interfacing and user-friendly operation can fulfill these requirements. In this Perspective article, the concept of an open platform for OoCs is defined from a technical perspective. Moreover, we discuss the importance of involving different stakeholders in the development, manufacturing, and application of such an open platform.

Microfluidic Electrochemistry Meets Trapped Ion Mobility Spectrometry and High-Resolution Mass Spectrometry-In Situ Generation, Separation, and Detection of Isomeric Conjugates of Paracetamol and Ethoxyquin.

Over the last 3 decades, electrochemistry (EC) has been successfully applied in phase I and phase II metabolism simulation studies. The electrochemically generated phase I metabolite-like oxidation products can react with selected reagents to form phase II conjugates. During conjugate formation, the generation of isomeric compounds is possible. Such isomeric conjugates are often separated by high-performance liquid chromatography (HPLC). Here, we demonstrate a powerful approach that combines EC with ion mobility spectrometry to separate possible isomeric conjugates. In detail, we present the hyphenation of a microfluidic electrochemical chip with an integrated mixer coupled online to trapped ion mobility spectrometry (TIMS) and time-of-flight high-resolution mass spectrometry (ToF-HRMS), briefly chipEC-TIMS-ToF-HRMS. This novel method achieves results in several minutes, which is much faster than traditional separation approaches like HPLC, and was applied to the drug paracetamol and the controversial feed preservative ethoxyquin. The analytes were oxidized in situ in the electrochemical microfluidic chip under formation of reactive intermediates and mixed with different thiol-containing reagents to form conjugates. These were analyzed by TIMS-ToF-HRMS to identify possible isomers. It was observed that the oxidation products of both paracetamol and ethoxyquin form two isomeric conjugates, which are characterized by different ion mobilities, with each reagent. Therefore, using this hyphenated technique, it is possible to not only form reactive oxidation products and their conjugates in situ but also separate and detect these isomeric conjugates within only a few minutes.

Autonomous capillary microfluidic devices with constant flow rate and temperature-controlled valving.

In this paper, we report on a capillary microfluidic device with constant flow rate and temperature-triggered stop valve function. It contains a PDMS channel that was grafted by a thermo-responsive polymer poly(N-isopropylacrylamide) (PNIPAm). The channel exhibits a constant capillary filling speed. By locally increasing the temperature in the channel from 20 °C to 37 °C using a microfabricated heater, a change of the surface wettability from hydrophilic to hydrophobic is obtained creating a hydrophobic stop valve. The valve can be reopened by lowering the temperature. The device is simple to fabricate and can be used as an actuatable capillary pump operating around room temperature. To understand the constant capillary filling speed, we performed contact angle measurements, in which we found slow wetting kinetics of PNIPAm-g-PDMS surfaces at temperatures below the lower critical solution temperature (LCST) of PNIPAm and fast wetting kinetics above the LCST. We interpret this as the result of the diffusive hydration process of PNIPAm below the LCST and the absence of hydration on the hydrophobic PNIPAm thin layer above the LCST.

Highly parallelized human embryonic stem cell differentiation to cardiac mesoderm in nanoliter chambers on a microfluidic chip.

Human stem cell-derived cells and tissues hold considerable potential for applications in regenerative medicine, disease modeling and drug discovery. The generation, culture and differentiation of stem cells in low-volume, automated and parallelized microfluidic chips hold great promise to accelerate the research in this domain. Here, we show that we can differentiate human embryonic stem cells (hESCs) to early cardiac mesodermal cells in microfluidic chambers that have a volume of only 30 nanoliters, using discontinuous medium perfusion. 64 of these chambers were parallelized on a chip which contained integrated valves to spatiotemporally isolate the chambers and automate cell culture medium exchanges. To confirm cell pluripotency, we tracked hESC proliferation and immunostained the cells for pluripotency markers SOX2 and OCT3/4. During differentiation, we investigated the effect of different medium perfusion frequencies on cell reorganization and the expression of the early cardiac mesoderm reporter MESP1mCherry by live-cell imaging. Our study demonstrates that microfluidic technology can be used to automatically culture, differentiate and study hESC in very low-volume culture chambers even without continuous medium perfusion. This result is an important step towards further automation and parallelization in stem cell technology.

Measuring barrier function in organ-on-chips with cleanroom-free integration of multiplexable electrodes.

Transepithelial/transendothelial electrical resistance (TEER) measurements can be applied in organ-on-chips (OoCs) to estimate the barrier properties of a tissue or cell layer in a continuous, non-invasive, and label-free manner. Assessing the barrier integrity in in vitro models is valuable for studying and developing barrier targeting drugs. Several systems for measuring the TEER have been shown, but each of them having their own drawbacks. This article presents a cleanroom-free fabrication method for the integration of platinum electrodes in a polydimethylsiloxane OoC, allowing the real-time assessment of the barrier function by employing impedance spectroscopy. The proposed method and electrode arrangement allow visual inspection of the cells cultured in the device at the site of the electrodes, and multiplexing of both the electrodes in one OoC and the number of OoCs in one device. The effectiveness of our system is demonstrated by lining the OoC with intestinal epithelial cells, creating a gut-on-chip, where we monitored the formation, as well as the disruption and recovery of the cell barrier during a 21 day culture period. The application is further expanded by creating a blood-brain-barrier, to show that the proposed fabrication method can be applied to monitor the barrier formation in the OoC for different types of biological barriers.

Single catalyst particle diagnostics in a microreactor for performing multiphase hydrogenation reactions.

Since inter- and intra-particle heterogeneities in catalyst particles are more the rule than the exception, it is advantageous to perform high-throughput screening for the activity of single catalyst particles. A multiphase system (gas/liquid/solid) is developed, where droplet-based microfluidics and optical detection are combined for the analysis of single catalyst particles by safely performing a hydrogenation study on in-house synthesized hollow Pd/SiO2 catalyst microparticles, in a polydimethylsiloxane (PDMS) microreactor. A two-phase segmented flow system of particle-containing droplets is combined with a parallel gas-reactant channel separated from the flow channel by a 50 µm thick gas permeable PDMS wall. In this paper, the developed microreactor system is showcased by monitoring the Pd-catalyzed hydrogenation of methylene blue. A discoloration of blue to brown visualizes the hydrogenation activity happening in a high-throughput fashion on the single Pd/SiO2 spherical catalyst microparticles, which are encapsulated in 50 nL-sized droplets. By measuring the reagent concentration at various spots along the length of the channel the reaction time can be determined, which is proportional to the residence time in the channel. The developed experimental platform opens new possibilities for single catalyst particle diagnostics in a multiphase environment.

Modular microreactor with integrated reflection element for online reaction monitoring using infrared spectroscopy.

We report on the fabrication of an internal reflection element (IRE) combined with a modular polymer microfluidic chip that can be used for attenuated total reflection (ATR) infrared spectroscopy. The IRE is fabricated from a silicon wafer. Two different polymers are used for the fabrication of the two types of modular microfluidic chips, namely polydimethylsiloxane (PDMS) and cyclic olefin copolymer (COC). The microfluidic chip is modular in the sense that several layers of mixing channels, using the herringbone mixer principle, and reactions chambers, can be stacked to facilitate the study of the desired reaction. A model Paal-Knorr reaction is carried out to prove that the chip works as intended. Furthermore, we highlight the strength of IR spectroscopy as a tool for reaction monitoring by identifying the peaks and showing the different reaction orders at the different steps of the Paal-Knorr reaction. The reduction of the aldehyde groups indicates a (pseudo) first order reaction whereas the vibrational modes associated with the ring formation indicate a zero order reaction. This zero order reaction can be explained with literature, where it is suggested that water acts as a catalyst during the dehydration step, which is the final step in the pyrrole ring formation.

Spectroelectrochemistry, the future of visualizing electrode processes by hyphenating electrochemistry with spectroscopic techniques.

The combination of electrochemistry and spectroscopy, known as spectroelectrochemistry (SEC), is an already established approach. By combining these two techniques, the relevance of the data obtained is greater than what it would be when using them independently. A number of review papers have been published on this subject, mostly written for experts in the field and focused on recent advances. In this review, written for both the novice in the field and the more experienced reader, the focus is not on the past but on the future. The scope is narrowed down to four techniques the authors claim to have the most potential for the future, namely: infrared spectroelectrochemistry (IR-SEC), Raman spectroelectrochemistry (Raman-SEC), nuclear magnetic resonance spectroelectrochemistry (NMR-SEC) and, perhaps slightly more controversial but certainly promising, electrochemistry mass-spectrometry (EC-MS).

Urea removal strategies for dialysate regeneration in a wearable artificial kidney.

The availability of a wearable artificial kidney (WAK) that provides dialysis outside the hospital would be an important advancement for dialysis patients. The concept of a WAK is based on regeneration of a small volume of dialysate in a closed-loop. Removal of urea, the primary waste product of nitrogen metabolism, is the major challenge for the realization of a WAK since it is a molecule with low reactivity that is difficult to adsorb while it is the waste solute with the highest daily molar production. Currently, no efficient urea removal technology is available that allows for miniaturization of the WAK to a size and weight that is acceptable for patients to carry. Several urea removal strategies have been explored, including enzymatic hydrolysis by urease, electro-oxidation and sorbent systems. However, thus far, these methods have toxic side effects, limited removal capacity or slow removal kinetics. This review discusses different urea removal strategies for application in a wearable dialysis device, from both a chemical and a medical perspective.

Wafer-scale fabrication of high-quality tunable gold nanogap arrays for surface-enhanced Raman scattering.

We report a robust and high-yield fabrication method for wafer-scale patterning of high-quality arrays of dense gold nanogaps, combining displacement Talbot lithography based shrink-etching with dry etching, wet etching, and thin film deposition techniques. By using the self-sharpening of <111>-oriented silicon crystal planes during the wet etching process, silicon structures with extremely smooth nanogaps are obtained. Subsequent conformal deposition of a silicon nitride layer and a gold layer results in dense arrays of narrow gold nanogaps. Using this method, we successfully fabricate high-quality Au nanogaps down to 10 nm over full wafer areas. Moreover, the gap spacing can be tuned by changing the thickness of deposited Au layers. Since the roughness of the template is minimized by the crystallographic etching of silicon, the roughness of the gold nanogaps depends almost exclusively on the roughness of the sputtered gold layers. Additionally, our fabricated Au nanogaps show a significant enhancement of surface-enhanced Raman scattering (SERS) signals of benzenethiol molecules chemisorbed on the structure surface, at an average enhancement factor up to 1.5 × 106.

A miniaturized push-pull-perfusion probe for few-second sampling of neurotransmitters in the mouse brain.

Measuring biomolecule concentrations in the brain of living animals, in real time, is a challenging task, especially when detailed information at high temporal resolution is also required. Traditionally, microdialysis probes are used that generally have sampling areas in the order of about 1 mm2, and provide information on concentrations with a temporal resolution of at least several minutes. In this paper, we present a novel miniaturized push-pull perfusion sampling probe that uses an array of small 3 µm-wide sampling channels to sample neurotransmitters at a typical recovery rate of 61%, with a reduced risk of clogging. The added feature to segment the dialysate inside the probe into small water-in-decane droplets enables the detection of concentrations with a temporal resolution of a few seconds. Here we used the probe for in vivo recordings of neurotransmitter glutamate released upon electrical stimulation in the brain of a mouse to demonstrate the feasibility of the probe for real-time neurochemical brain analysis.

Luminescence thermometry for in situ temperature measurements in microfluidic devices.

Temperature control for lab-on-a-chip devices has resulted in the broad applicability of microfluidics to, e.g., polymerase chain reaction (PCR), temperature gradient focusing for electrophoresis, and colloidal particle synthesis. However, currently temperature sensors on microfluidic chips either probe temperatures outside the channel (resistance temperature detector, RTD) or are limited in both the temperature range and sensitivity in the case of organic dyes. In this work, we introduce ratiometric bandshape luminescence thermometry in which thermally coupled levels of Er3+ in NaYF4 nanoparticles are used as a promising method for in situ temperature mapping in microfluidic systems. The results, obtained with three types of microfluidic devices, demonstrate that temperature can be monitored inside a microfluidic channel accurately (0.34 °C) up to at least 120 °C with a spot size of ca. 1 mm using simple fiber optics. Higher spatial resolution can be realized by combining luminescence thermometry with confocal microscopy, resulting in a spot size of ca. 9 µm. Further improvement is anticipated to enhance the spatial resolution and allow for 3D temperature profiling.

Non-invasive sensing of transepithelial barrier function and tissue differentiation in organs-on-chips using impedance spectroscopy.

Here, we describe methods for combining impedance spectroscopy measurements with electrical simulation to reveal transepithelial barrier function and tissue structure of human intestinal epithelium cultured inside an organ-on-chip microfluidic culture device. When performing impedance spectroscopy measurements, electrical simulation enabled normalization of cell layer resistance of epithelium cultured statically in a gut-on-a-chip, which enabled determination of transepithelial electrical resistance (TEER) values that can be compared across device platforms. During culture under dynamic flow, the formation of intestinal villi was accompanied by characteristic changes in impedance spectra both measured experimentally and verified with simulation, and we demonstrate that changes in cell layer capacitance may serve as measures of villi differentiation. This method for combining impedance spectroscopy with simulation can be adapted to better monitor cell layer characteristics within any organ-on-chip in vitro and to enable direct quantitative TEER comparisons between organ-on-chip platforms which should help to advance research on organ function.