The Importance of Dean Flow in Microfluidic Nanoparticle Synthesis: A ZIF-8 Case Study.

The Dean Flow, a physics phenomenon that accounts for the impact of channel curvature on fluid dynamics, has great potential to be used in microfluidic synthesis of nanoparticles. This study explores the impact of the Dean Flow on the synthesis of ZIF-8 particles. Several variables that influence the Dean Equation (the mathematical expression of Dean Flow) are tested to validate the applicability of this expression in microfluidic synthesis, including the flow rate, radius of curvature, channel cross sectional area, and reagent concentration. It is demonstrated that the current standard of reporting, providing only the flow rate and crucially not the radius of curvature, is an incomplete description that will invariably lead to irreproducible syntheses across different laboratories. An alternative standard of reporting is presented and it is demonstrated how the sleek and simple math of the Dean Equation can be used to precisely tune the final dimensions of high quality, monodisperse ZIF-8 nanoparticles between 40 and 700 nm.

Nanofluidic Immobilization and Growth Detection of Escherichia coli in a Chip for Antibiotic Susceptibility Testing.

Infections with antimicrobial resistant bacteria are a rising threat for global healthcare as more and more antibiotics lose their effectiveness against bacterial pathogens. To guarantee the long-term effectiveness of broad-spectrum antibiotics, they may only be prescribed when inevitably required. In order to make a reliable assessment of which antibiotics are effective, rapid point-of-care tests are needed. This can be achieved with fast phenotypic microfluidic tests, which can cope with low bacterial concentrations and work label-free. Here, we present a novel optofluidic chip with a cross-flow immobilization principle using a regular array of nanogaps to concentrate bacteria and detect their growth label-free under the influence of antibiotics. The interferometric measuring principle enabled the detection of the growth of Escherichia coli in under 4 h with a sample volume of 187.2 µL and a doubling time of 79 min. In proof-of-concept experiments, we could show that the method can distinguish between bacterial growth and its inhibition by antibiotics. The results indicate that the nanofluidic chip approach provides a very promising concept for future rapid and label-free antimicrobial susceptibility tests.

In situ Microfluidic Cryofixation for Cryo Focused Ion Beam Milling and Cryo Electron Tomography.

We present a microfluidic platform for studying structure-function relationships at the cellular level by connecting video rate live cell imaging with in situ microfluidic cryofixation and cryo-electron tomography of near natively preserved, unstained specimens. Correlative light and electron microscopy (CLEM) has been limited by the time required to transfer live cells from the light microscope to dedicated cryofixation instruments, such as a plunge freezer or high-pressure freezer. We recently demonstrated a microfluidic based approach that enables sample cryofixation directly in the light microscope with millisecond time resolution, a speed improvement of up to three orders of magnitude. Here we show that this cryofixation method can be combined with cryo-electron tomography (cryo-ET) by using Focused Ion Beam milling at cryogenic temperatures (cryo-FIB) to prepare frozen hydrated electron transparent sections. To make cryo-FIB sectioning of rapidly frozen microfluidic channels achievable, we developed a sacrificial layer technique to fabricate microfluidic devices with a PDMS bottom wall <5 µm thick. We demonstrate the complete workflow by rapidly cryo-freezing Caenorhabditis elegans roundworms L1 larvae during live imaging in the light microscope, followed by cryo-FIB milling and lift out to produce thin, electron transparent sections for cryo-ET imaging. Cryo-ET analysis of initial results show that the structural preservation of the cryofixed C. elegans was suitable for high resolution cryo-ET work. The combination of cryofixation during live imaging enabled by microfluidic cryofixation with the molecular resolution capabilities of cryo-ET offers an exciting avenue to further advance space-time correlative light and electron microscopy (st-CLEM) for investigation of biological processes at high resolution in four dimensions.

Nanoparticle Characterization: What to Measure?

What to measure? is a key question in nanoscience, and it is not straightforward to address as different physicochemical properties define a nanoparticle sample. Most prominent among these properties are size, shape, surface charge, and porosity. Today researchers have an unprecedented variety of measurement techniques at their disposal to assign precise numerical values to those parameters. However, methods based on different physical principles probe different aspects, not only of the particles themselves, but also of their preparation history and their environment at the time of measurement. Understanding these connections can be of great value for interpreting characterization results and ultimately controlling the nanoparticle structure-function relationship. Here, the current techniques that enable the precise measurement of these fundamental nanoparticle properties are presented and their practical advantages and disadvantages are discussed. Some recommendations of how the physicochemical parameters of nanoparticles should be investigated and how to fully characterize these properties in different environments according to the intended nanoparticle use are proposed. The intention is to improve comparability of nanoparticle properties and performance to ensure the successful transfer of scientific knowledge to industrial real-world applications.

Robust Smartphone Assisted Biosensing Based on Asymmetric Nanofluidic Grating Interferometry.

Point-of-care systems enable fast therapy decisions on site without the need of any healthcare infrastructure. In addition to the sensitive detection, stable measurement by inexperienced persons outside of laboratory facilities is indispensable. A particular challenge in field applications is to reduce interference from environmental factors, such as temperature, to acceptable levels without sacrificing simplicity. Here, we present a smartphone-based point-of-care sensor. The method uses an optofluidic grating composed of alternating detection and reference channels arranged as a reflective phase grating. Biomolecules adsorbing to the detection channel alter the optical path length, while the parallel reference channels enable a direct common mode rejection within a single measurement. The optical setup is integrated in a compact design of a mobile readout device and the usability is ensured by a smartphone application. Our results show that different ambient temperatures do not have any influence on the signal. In a proof-of concept experiment we measured the accumulation of specific molecules in functionalized detection channels in real-time and without the need of any labeling. Therefore, the channel walls have been modified with biotin as capture molecules and the specific binding of streptavidin was detected. A mobile, reliable and robust point-of-care device has been realized by combining an inherently differential measurement concept with a smartphone-based, mobile readout device.

A Microfluidic Split-Flow Technology for Product Characterization in Continuous Low-Volume Nanoparticle Synthesis.

A key aspect of microfluidic processes is their ability to perform chemical reactions in small volumes under continuous flow. However, a continuous process requires stable reagent flow over a prolonged period. This can be challenging in microfluidic systems, as bubbles or particles easily block or alter the flow. Online analysis of the product stream can alleviate this problem by providing a feedback signal. When this signal exceeds a pre-defined range, the process can be re-adjusted or interrupted to prevent contamination. Here we demonstrate the feasibility of this concept by implementing a microfluidic detector downstream of a segmented-flow system for the synthesis of lipid nanoparticles. To match the flow rate through the detector to the measurement bandwidth independent of the synthesis requirements, a small stream is sidelined from the original product stream and routed through a measuring channel with 2 × 2 µm cross-section. The small size of the measuring channel prevents the entry of air plugs, which are inherent to our segmented flow synthesis device. Nanoparticles passing through the small channel were detected and characterized by quantitative fluorescence measurements. With this setup, we were able to count single nanoparticles. This way, we were able to detect changes in the particle synthesis affecting the size, concentration, or velocity of the particles in suspension. We envision that the flow-splitting scheme demonstrated here can be transferred to detection methods other than fluorescence for continuous monitoring and feedback control of microfluidic nanoparticle synthesis.

Mass Measurements Reveal Preferential Sorption of Mixed Solvent Components in Porous Nanoparticles.

The interplay of physical and chemical properties at the nanometer scale provides porous nanoparticles with unique sorption and interaction capabilities. These properties have aroused great interest toward this class of materials for application ranging from chemical and biological sensing to separation and drug delivery. However, so far the preferential uptake of different components of mixed solvents by porous nanoparticles is not measured due to a lack of methods capable of detecting the resulting change in physical properties. Here, a new method, nanomechanical mass correlation spectroscopy, is used to reveal an unexpected dependence of the effective mass density of porous metal-organic framework (MOF) nanoparticles on the chemistry of the solvent system and on the chemical functionalization of the MOF's internal surface. Interestingly, the pore size of the nanoparticles is much too large for the exclusion of small solvent molecules by steric hindrance. The variation of effective density of the nanoparticles with the solvent composition indicates that a complex solvent environment can form within or around the nanoparticles, which may substantially differ from the solvent composition.

Aberration-corrected cryoimmersion light microscopy.

Cryogenic fluorescent light microscopy of flash-frozen cells stands out by artifact-free fixation and very little photobleaching of the fluorophores used. To attain the highest level of resolution, aberration-free immersion objectives with accurately matched immersion media are required, but both do not exist for imaging below the glass-transition temperature of water. Here, we resolve this challenge by combining a cryoimmersion medium, HFE-7200, which matches the refractive index of room-temperature water, with a technological concept in which the body of the objective and the front lens are not in thermal equilibrium. We implemented this concept by replacing the metallic front-lens mount of a standard bioimaging water immersion objective with an insulating ceramic mount heated around its perimeter. In this way, the objective metal housing can be maintained at room temperature, while creating a thermally shielded cold microenvironment around the sample and front lens. To demonstrate the range of potential applications, we show that our method can provide superior contrast in Escherichia coli and yeast cells expressing fluorescent proteins and resolve submicrometer structures in multicolor immunolabeled human bone osteosarcoma epithelial (U2OS) cells at [Formula: see text]C.

Continuous high throughput nanofluidic separation through tangential-flow vertical nanoslit arrays.

Nanofluidic devices exhibit unique, tunable transport properties that may lead to breakthroughs in molecular separations and sensing. However, the throughput of these devices is orders of magnitude too small for the processing of macroscopic samples. Here we overcome this problem by combining two technological innovations. First, nanofluidic channels are made as vertical slits connecting the two sides of a silicon nitride membrane. Arbitrary arrays of such nanoslits down to 15 nm wide with <6 Å uniformity were made by merging the idea of templating with chemical mechanical polishing to create a scalable, nanolithography-free wafer level process. Second, we provide for efficient solute transport to and from the openings of the nanoslits by incorporating the nanofluidic membrane into a microfluidic tangential-flow system, which is also fabricated at wafer level. As an exemplary application, we demonstrate charge-based continuous flow separation of small molecules with a selectivity of 100 and constant flux over more than 100 hours of operation. This proves the exciting possibility of exploiting transport phenomena governed by precision-engineered nanofluidic devices at a macroscopic scale.

Microfluidic cryofixation for correlative microscopy.

Cryofixation yields outstanding ultrastructural preservation of cells for electron microscopy, but current methods disrupt live cell imaging. Here we demonstrate a microfluidic approach that enables cryofixation to be performed directly in the light microscope with millisecond time resolution and at atmospheric pressure. This will provide a link between imaging/stimulation of live cells and post-fixation optical, electron, or X-ray microscopy.

Resolution enhancement of suspended microchannel resonators for weighing of biomolecular complexes in solution.

We introduce the use of correlation analysis to extend the dynamic range of suspended micro- and nanochannel resonator (SMR/SNR) mass sensors by over five orders of magnitude. This method can analyze populations of particles flowing through an embedded channel micromechanical resonator, even when the individual particle masses are far below the noise floor. To characterize the method, we measured the mass of polystyrene nanoparticles with 300 zg resolution. As an application, we monitored the time course of insulin amyloid formation from pre-fibrillar aggregates to mature fibrils of 15 MDa average mass. Results were compared with thioflavin-T (ThT) assays and electron microscopy (EM). Mass measurements offer additional information over ThT during the fluorescent inaccessible lag period, and the average fibril dimensions calculated from the mass signal are in good accordance with EM. In the future, we envision that more detailed modeling will allow the computational deconvolution of multicomponent samples, enabling the mass spectrometric characterization of a variety of biomolecular complexes, small organelles, and nanoparticles in solution.

A fluorescence based method for the quantification of surface functional groups in closed micro- and nanofluidic channels.

Surface analysis is critical for the validation of microfluidic surface modifications for biology, chemistry, and physics applications. However, until now quantitative analytical methods have mostly been focused on open surfaces. Here, we present a new fluorescence imaging method to directly measure the surface coverage of functional groups inside assembled microchannels over a wide dynamic range. A key advance of our work is the elimination of self-quenching to obtain a linear signal even with a high density of functional groups. This method is applied to image the density and monitor the stability of vapor deposited silane layers in bonded silicon/glass micro- and nanochannels.

Mass sensors with mechanical traps for weighing single cells in different fluids.

We present two methods by which single cells can be mechanically trapped and continuously monitored within the suspended microchannel resonator (SMR) mass sensor. Since the fluid surrounding the trapped cell can be quickly and completely replaced on demand, our methods are well suited for measuring changes in cell size and growth in response to drugs or other chemical stimuli. We validate our methods by measuring the density of single polystyrene beads and Saccharomyces cerevisiae yeast cells with a precision of approximately 10(-3) g cm(-3), and by monitoring the growth of single mouse lymphoblast cells before and after drug treatment.

Energy dissipation in microfluidic beam resonators: effect of Poisson's ratio.

Dissipation of mechanical energy underlies the sensitivity of many nanomechanical devices, with environmental effects often having a significant effect. One case of practical relevance is the interaction of elastic beam resonators with fluid, which is known to dramatically increase energy dissipation. Recently, we investigated energy dissipation in a different class of elastic beam resonator that embeds a microfluidic channel in its interior. In this paper, we examine the effect of the beam material Poisson ratio on these devices and discover that it can strongly affect energy dissipation--this is in direct contrast to conventional cantilever beams immersed in fluid. Increasing the Poisson ratio in these microfluidic devices is found to decrease energy dissipation, with the incompressible material limit providing minimum energy dissipation. Our paper establishes that, in this limit, placement of the fluid channel away from the beam neutral axis has negligible effect on energy dissipation in many cases of practical interest. The physical implications of these findings are discussed, and a detailed comparison with available experimental results is provided.

Toward attogram mass measurements in solution with suspended nanochannel resonators.

Using suspended nanochannel resonators (SNRs), we demonstrate measurements of mass in solution with a resolution of 27 ag in a 1 kHz bandwidth, which represents a 100-fold improvement over existing suspended microchannel resonators and, to our knowledge, is the most precise mass measurement in liquid today. The SNR consists of a cantilever that is 50 microm long, 10 microm wide, and 1.3 microm thick, with an embedded nanochannel that is 2 microm wide and 700 nm tall. The SNR has a resonance frequency near 630 kHz and exhibits a quality factor of approximately 8000 when dry and when filled with water. In addition, we introduce a new method that uses centrifugal force caused by vibration of the cantilever to trap particles at the free end. This approach eliminates the intrinsic position dependent error of the SNR and also improves the mass resolution by increasing the averaging time for each particle.

Nonmonotonic energy dissipation in microfluidic resonators.

Nanomechanical resonators enable a range of precision measurements in air or vacuum, but strong viscous damping makes applications in liquid challenging. Recent experiments have shown that fluid damping is greatly reduced in fluidic embedded-channel microcantilevers. Here we report the discovery of nonmonotonic energy dissipation due to the fluid in such devices, which leads to the intriguing prospect of enhancing the quality factor upon miniaturization. These observations elucidate the physical mechanisms of energy dissipation in embedded-channel resonators and thus provide the basis for numerous applications in nanoscience and biology.

Integrated measurement of the mass and surface charge of discrete microparticles using a suspended microchannel resonator.

Measurements of the mass and surface charge of microparticles are employed in the characterization of many types of colloidal dispersions. The suspended microchannel resonator (SMR) is capable of measuring individual particle masses with femtogram resolution. Here, we employ the high sensitivity of the SMR resonance frequency to changes in particle position, relative to the cantilever tip, to determine the electrophoretic mobility of discrete particles in an applied electric field. When a sinusoidal electric field is applied to the suspended microchannel, the transient resonance frequency shift corresponding to a particle transit can be analyzed by digital signal processing to extract both the buoyant mass and electrophoretic mobility of each particle. These parameters, together with the mean particle density, can be used to compute the size, absolute mass, and surface charge of discrete microspheres, leading to a true representation of the mean and polydispersity of these quantities for a population. We have applied this technique to an aqueous suspension of two types of polystyrene microspheres, to differentiate them based on their absolute mass and their surface charge. The integrated measurement of electrophoretic mobility using the SMR is determined to be quantitative, based on comparison with commercial instruments, and exhibits favorable scaling properties that will ultimately enable measurements from mammalian cells.

Suspended microchannel resonators for ultralow volume universal detection.

Universal detectors that maintain high sensitivity as the detection volume is reduced to the subnanoliter scale can enhance the utility of miniaturized total analysis systems (mu-TAS). Here the unique scaling properties of the suspended microchannel resonator (SMR) are exploited to show universal detection in a 10 pL analysis volume with a density detection limit of approximately 1 microg/cm (3) (10 Hz bandwidth) and a dynamic range of 6 decades. Analytes with low UV extinction coefficients such as polyethylene glycol (PEG) 8 kDa, glucose, and glycine are measured with molar detection limits of 0.66, 13.5, and 31.6 microM, respectively. To demonstrate the potential for real-time monitoring, gel filtration chromatography was used to separate different molecular weights of PEG as the SMR acquired a chromatogram by measuring the eluate density. This work suggests that the SMR could offer a simple and sensitive universal detector for various separation systems from liquid chromatography to capillary electrophoresis. Moreover, since the SMR is itself a microfluidic channel, it can be directly integrated into mu-TAS without compromising overall performance.

Weighing of biomolecules, single cells and single nanoparticles in fluid.

Nanomechanical resonators enable the measurement of mass with extraordinary sensitivity. Previously, samples as light as 7 zeptograms (1 zg = 10(-21) g) have been weighed in vacuum, and proton-level resolution seems to be within reach. Resolving small mass changes requires the resonator to be light and to ring at a very pure tone-that is, with a high quality factor. In solution, viscosity severely degrades both of these characteristics, thus preventing many applications in nanotechnology and the life sciences where fluid is required. Although the resonant structure can be designed to minimize viscous loss, resolution is still substantially degraded when compared to measurements made in air or vacuum. An entirely different approach eliminates viscous damping by placing the solution inside a hollow resonator that is surrounded by vacuum. Here we demonstrate that suspended microchannel resonators can weigh single nanoparticles, single bacterial cells and sub-monolayers of adsorbed proteins in water with sub-femtogram resolution (1 Hz bandwidth). Central to these results is our observation that viscous loss due to the fluid is negligible compared to the intrinsic damping of our silicon crystal resonator. The combination of the low resonator mass (100 ng) and high quality factor (15,000) enables an improvement in mass resolution of six orders of magnitude over a high-end commercial quartz crystal microbalance. This gives access to intriguing applications, such as mass-based flow cytometry, the direct detection of pathogens, or the non-optical sizing and mass density measurement of colloidal particles.

Transient signal analysis using complementary metal oxide semiconductor capacitive chemical microsensors.

This work explores the possibility to discriminate analytes based on their nonequilibrium signals in polymer-coated capacitive chemical microsensors. The analyte uptake in the chemically sensitive polymer layers of 3-7-microm thickness has been analyzed using a diffusion model and the dynamic sensor response data. The shapes of the response profiles have been calculated analytically. Despite the simplifications in the model, the observed transient signal profiles could be described accurately. Comparison of the measured diffusion coefficients (on the order of 10(-12) m2/s) with literature values measured at similar concentration levels showed good agreement. Concentration-independent diffusion coefficients for several analyte/polymer combinations (poly(etherurethane)/all analytes; poly(epichlorohydrin)/alcohols) as well as slightly concentration-dependent diffusion coefficients (poly(epichlorohydrin)/toluene or ethyl cellulose/toluene) have been found in the investigated concentration range of tens to hundreds of pascals gas-phase partial pressure. The diffusion times of water and the first aliphatic monohydric alcohols in the polymers are strongly correlated to their molecular size. The discrimination of these substances based on dynamic sensor data of a single sensor could be demonstrated. In particular, the analysis of mixtures of analytes with similar chemical behavior (water/ethanol or methanol/ethanol) by means of analyzing the response profile of single-exposure steps or by applying a series of decreasingly long alternating target gas exposure and carrier gas exposure steps has been performed.