Geometry and length control of 3D engineered heart tissues using direct laser writing.

Geometry and mechanical characteristics of the environment surrounding the Engineered Heart Tissues (EHT) affect their structure and function. Here, we employed a 3D tissue culture platform fabricated using two-photon direct laser writing with a high degree of accuracy to control parameters that are relevant to EHT maturation. Using this platform, we first explore the effects of geometry based on two distinct shapes: a rectangular seeding well with two attachment sites, and a stadium-like seeding well with six attachment sites that are placed symmetrically along hemicylindrical membranes. The former geometry promotes uniaxial contraction of the tissues; the latter additionally induces diagonal fiber alignment. We systematically increase the length of the seeding wells for both configurations and observe a positive correlation between fiber alignment at the center of the EHTs and tissue length. With increasing length, an undesirable thinning and "necking" also emerge, leading to the failure of longer tissues over time. In the second step, we optimize the stiffness of the seeding wells and modify some of the attachment sites of the platform and the seeding parameters to achieve tissue stability for each length and geometry. Furthermore, we use the platform for electrical pacing and calcium imaging to evaluate the functional dynamics of EHTs as a function of frequency.

Measurement of the low-frequency charge noise of bacteria.

Bacteria meticulously regulate their intracellular ion concentrations and create ionic concentration gradients across the bacterial membrane. These ionic concentration gradients provide free energy for many cellular processes and are maintained by transmembrane transport. Given the physical dimensions of a bacterium and the stochasticity in transmembrane transport, intracellular ion concentrations and hence the charge state of a bacterium are bound to fluctuate. Here we investigate the charge noise of hundreds of nonmotile bacteria by combining electrical measurement techniques from condensed matter physics with microfluidics. In our experiments, bacteria in a microchannel generate charge density fluctuations in the embedding electrolyte due to random influx and efflux of ions. Detected as electrical resistance noise, these charge density fluctuations display a power spectral density proportional to 1/f^{2} for frequencies 0.05Hz≤f≤1Hz. Fits to a simple noise model suggest that the steady-state charge of a bacterium fluctuates by ±1.30×10^{6}e(e≈1.60×10^{-19}C), indicating that bacterial ion homeostasis is highly dynamic and dominated by strong charge noise. The rms charge noise can then be used to estimate the fluctuations in the membrane potential; however, the estimates are unreliable due to our limited understanding of the intracellular concentration gradients.

Frequency-Dependent Piezoresistive Effect in Top-down Fabricated Gold Nanoresistors.

Piezoresistive strain gauges allow for electronic readout of mechanical deformations with high fidelity. As piezoresistive strain gauges are aggressively being scaled down for applications in nanotechnology, it has become critical to investigate their physical attributes at different limits. Here, we describe an experimental approach for studying the piezoresistive gauge factor of a gold thin-film nanoresistor as a function of frequency. The nanoresistor is fabricated lithographically near the anchor of a nanomechanical doubly clamped beam resonator. As the resonator is driven to resonance in one of its normal modes, the nanoresistor is exposed to frequency-dependent strains of ε ≲ 10-5 in the 4-36 MHz range. We calibrate the strain using optical interferometry and measure the resistance changes using a radio frequency mix-down technique. The piezoresistive gauge factor γ of our lithographic gold nanoresistors is γ ≈ 3.6 at 4 MHz, in agreement with comparable macroscopic thin metal film resistors in previous works. However, our γ values increase monotonically with frequency and reach γ ≈ 15 at 36 MHz. We discuss possible physics that may give rise to this unexpected frequency dependence.

Direct laser writing for cardiac tissue engineering: a microfluidic heart on a chip with integrated transducers.

We have developed a microfluidic platform for engineering cardiac microtissues in highly-controlled microenvironments. The platform is fabricated using direct laser writing (DLW) lithography and soft lithography, and contains four separate devices. Each individual device houses a cardiac microtissue and is equipped with an integrated strain actuator and a force sensor. Application of external pressure waves to the platform results in controllable time-dependent forces on the microtissues. Conversely, oscillatory forces generated by the microtissues are transduced into measurable electrical outputs. We demonstrate the capabilities of this platform by studying the response of cardiac microtissues derived from human induced pluripotent stem cells (hiPSC) under prescribed mechanical loading and pacing. This platform will be used for fundamental studies and drug screening on cardiac microtissues.

Nanomechanical Measurement of the Brownian Force Noise in a Viscous Liquid.

We study the frequency spectrum of the thermal force giving rise to Brownian motion of a nanomechanical beam resonator in a viscous liquid. In the first set of experiments, we measure the power spectral density (PSD) of the position fluctuations of the resonator around its fundamental mode at its center. Then, we measure the frequency-dependent linear response of the resonator, again at its center, by driving it with a harmonic force that couples well to the fundamental mode. These two measurements allow us to determine the PSD of the Brownian force noise acting on the structure in its fundamental mode. The PSD of the force noise from multiple resonators spanning a broad frequency range displays a "colored spectrum" and follows the dissipation of a blade oscillating in a viscous liquid-by virtue of the fluctuation-dissipation theorem of statistical mechanics.

Matrix Pore Size Governs Escape of Human Breast Cancer Cells from a Microtumor to an Empty Cavity.

How the extracellular matrix (ECM) affects the progression of a localized tumor to invasion of the ECM and eventually to vascular dissemination remains unclear. Although many studies have examined the role of the ECM in early stages of tumor progression, few have considered the subsequent stages that culminate in intravasation. In the current study, we have developed a three-dimensional (3D) microfluidic culture system that captures the entire process of invasion from an engineered human micro-tumor of MDA-MB-231 breast cancer cells through a type I collagen matrix and escape into a lymphatic-like cavity. By varying the physical properties of the collagen, we have found that MDA-MB-231 tumor cells invade and escape faster in lower-density ECM. These effects are mediated by the ECM pore size, rather than by the elastic modulus or interstitial flow speed. Our results underscore the importance of ECM structure in the vascular escape of human breast cancer cells.

All-electrical monitoring of bacterial antibiotic susceptibility in a microfluidic device.

The lack of rapid antibiotic susceptibility tests adversely affects the treatment of bacterial infections and contributes to increased prevalence of multidrug-resistant bacteria. Here, we describe an all-electrical approach that allows for ultrasensitive measurement of growth signals from only tens of bacteria in a microfluidic device. Our device is essentially a set of microfluidic channels, each with a nanoconstriction at one end and cross-sectional dimensions close to that of a single bacterium. Flowing a liquid bacteria sample (e.g., urine) through the microchannels rapidly traps the bacteria in the device, allowing for subsequent incubation in drugs. We measure the electrical resistance of the microchannels, which increases (or decreases) in proportion to the number of bacteria in the microchannels. The method and device allow for rapid antibiotic susceptibility tests in about 2 h. Further, the short-time fluctuations in the electrical resistance during an antibiotic susceptibility test are correlated with the morphological changes of bacteria caused by the antibiotic. In contrast to other electrical approaches, the underlying geometric blockage effect provides a robust and sensitive signal, which is straightforward to interpret without electrical models. The approach also obviates the need for a high-resolution microscope and other complex equipment, making it potentially usable in resource-limited settings.

Characterization and manipulation of single nanoparticles using a nanopore-based electrokinetic tweezer.

Manipulation and characterization of nanoscale objects through electrokinetic techniques offer numerous advantages compared to the existing optical methods and hold great potential for both fundamental research and practical applications. Here we present a novel electrokinetic tweezer for single nanoparticle manipulation and characterization based on electrokinetic trapping near a low-aspect-ratio nanopore. We find that this nanopore-based electrokinetic tweezer share lots of similarity with optical tweezers and can be modeled as an overdamped harmonic oscillator, with the spring constant of the system being the trap stiffness. We show that different values of ionic currents through the nanopore and trap stiffnesses are achieved when trapping nanoparticles with different sizes (down to 100 nm) and/or zeta potentials. We also demonstrate that the trap stiffness and nanoparticle position can be easily tuned by changing the applied voltage and buffer concentration. We envision that further development of this electrokinetic tweezer will enable various advanced tools for nanophotonics, drug delivery, and biosensing.

Microfluidic detection of movements of Escherichia coli for rapid antibiotic susceptibility testing.

Various nanomechanical movements of bacteria provide a signature of bacterial viability. Most notably, bacterial movements have been observed to subside rapidly and dramatically when the bacteria are exposed to effective antibiotics. Thus, monitoring bacterial movements, if performed with high fidelity, could offer a path to various clinical microbiological applications, including antibiotic susceptibility tests. Here, we introduce a robust and ultrasensitive electrical transduction technique for detecting the nanomechanical movements of bacteria. The technique is based on measuring the electrical fluctuations in a microfluidic channel, which the bacteria populate. The swimming of planktonic bacteria and the random oscillations of surface-immobilized bacteria both cause small but detectable electrical fluctuations. We show that this technique provides enough sensitivity to detect even the slightest movements of a single cell; we also demonstrate an antibiotic susceptibility test in a biological matrix. Given that it lends itself to smooth integration with other microfluidic methods and devices, the technique can be developed into a functional antibiotic susceptibility test, in particular, for urinary tract infections.

Dynamics of Interstitial Fluid Pressure in Extracellular Matrix Hydrogels in Microfluidic Devices.

In order to understand how interstitial fluid pressure and flow affect cell behavior, many studies use microfluidic approaches to apply externally controlled pressures to the boundary of a cell-containing gel. It is generally assumed that the resulting interstitial pressure distribution quickly reaches a steady-state, but this assumption has not been rigorously tested. Here, we demonstrate experimentally and computationally that the interstitial fluid pressure within an extracellular matrix gel in a microfluidic device can, in some cases, react with a long time delay to external loading. Remarkably, the source of this delay is the slight (∼100 nm in the cases examined here) distension of the walls of the device under pressure. Finite-element models show that the dynamics of interstitial pressure can be described as an instantaneous jump, followed by axial and transverse diffusion, until the steady pressure distribution is reached. The dynamics follow scaling laws that enable estimation of a gel's poroelastic constants from time-resolved measurements of interstitial fluid pressure.

Non-invasive mapping of interstitial fluid pressure in microscale tissues.

This study describes a non-invasive method for mapping interstitial fluid pressure within hydrogel-based microscale tissues. The method is based on embedding (or forming) a tissue within a silicone (PDMS) microfluidic device, and measuring the extremely slight displacement (<1 µm) of the PDMS optically when the device is pressurized under static and flow conditions. The displacement field under uniform pressure provides a map of the local device stiffness, which can then be used to obtain the non-uniform pressure field under flow conditions. We have validated this method numerically and applied it towards determining the hydraulic properties of tumor cell aggregates, blind-ended epithelial tubes, and perfused endothelial tubes that were all cultured within micropatterned collagen gels. The method provides an accessible tool for generating high-resolution maps of interstitial fluid pressure for studies in mechanobiology.

Monolithic integration of a nanomechanical resonator to an optical microdisk cavity.

We report a Silicon nano-opto-mechanical device in which a nanomechanical doubly-clamped beam resonator is integrated to an optical microdisk cavity. Small flexural oscillations of the beam cause intensity modulations in the circulating optical field in the nearby microdisk cavity. By monitoring the corresponding fluctuations in the cavity transmission via a fiber-taper, one can detect these oscillations with a displacement sensitivity approaching 10 fm·Hz-1/2 at an input power level of 50 µW. Both the in-plane and out-of-plane fundamental flexural resonances of the beam can be read out by this approach - the latter being detectable due to broken planar symmetry in the system. Access to multiple mechanical modes of the same resonator may be useful in some applications and may enable interesting fundamental studies.

Sensitive micromechanical displacement detection by scattering evanescent optical waves.

We describe a simple approach to detect small mechanical displacements by scattering evanescent optical waves confined around an optical waveguide. Our experimental setup consists of a microcantilever brought into the proximity of a tapered optical fiber. The scattering of evanescent waves and hence the optical transmission through the tapered fiber is strongly dependent on the separation between the fiber and the microcantilever, allowing for sensitive detection of the small oscillations of the microcantilever. Our approach does not require a coherent laser source, yet it provides a displacement sensitivity of ~260fmHz(-1/2) at a small power level of 38microW. It is suitable for scanning probe microscopy and could eventually be adapted to nanomechanical resonators.

Diffraction of evanescent waves and nanomechanical displacement detection.

Sensitive displacement detection has emerged as a significant technological challenge in mechanical resonators with nanometer-scale dimensions. A novel nanomechanical displacement detection scheme based upon the scattering of focused evanescent fields is proposed. The sensitivity of the proposed approach is studied using diffraction theory of evanescent waves. Diffraction theory results are compared with numerical simulations.

Efficient and sensitive capacitive readout of nanomechanical resonator arrays.

Here we describe all-electronic broadband motion detection in radio frequency nanomechanical resonators. Our technique relies upon the measurement of small motional capacitance changes using an LC impedance transformation network. We first demonstrate the technique on a single doubly clamped beam resonator with a side gate over a wide range of temperatures from 20 mK to 300 K. We then apply the technique to accomplish multiplexed readout of an array of individually addressable resonators, all embedded in a single high-frequency circuit. This technique may find use in a variety of applications ranging from ultrasensitive mass and force sensing to quantum information processing.