Digital nucleic acid amplification testing (dNAAT) and analysis techniques, such as digital polymerase chain reaction (PCR), have become useful clinical diagnostic tools. However, nucleic acid (NA) sample preparation preceding dNAAT is generally laborious and performed manually, thus creating the need for a simple sample preparation technique and a facile coupling strategy for dNAAT. Therefore, we demonstrate a simple workflow which automates magnetic bead-based extraction of NAs with a one-step transfer to dNAAT. Specifically, we leverage droplet magnetofluidics (DM) to automate the movement of magnetic beads between small volumes of reagents commonly employed for NA extraction and purification. Importantly, the buffer typically used to elute the NAs off the magnetic beads is replaced by a carefully selected PCR solution, enabling direct transfer from sample preparation to dNAAT. Moreover, we demonstrate the potential for multiplexing using a digital high-resolution melt (dHRM) after the digital PCR (dPCR). The utility of this workflow is demonstrated with duplexed detection of bacteria in a sample imitating a coinfection. We first purify the bacterial DNA into a PCR solution using our DM-based sample preparation. We then transfer the purified bacterial DNA to our microfluidic nanoarray to amplify 16S rRNA using dPCR and then perform dHRM to identify the two bacterial species.

Sprays are a class of multiphase flows which exhibit a wide range of drop size and velocity scales spanning several orders of magnitude. The objective of the current work is to experimentally investigate the prospect of dynamical similarity in these flows. We are also motivated to identify a choice of length and time scales which could lead towards a universal description of the drop size and velocity spectra. Towards this end, we have fabricated a cohort of geometrically similar pressure swirl atomizers using micro-electromechanical systems (MEMS) as well as additive manufacturing technology. We have characterized the dynamical characteristics of the sprays as well as the drop size and velocity spectra (in terms of probability density functions, p.d.f.s) over a wide range of Reynolds ($Re$) and Weber numbers ($We$) using high-speed imaging and phase Doppler interferometry, respectively. We show that the dimensionless Sauter mean diameter ($D_{32}$) scaled to the boundary layer thickness in the liquid sheet at the nozzle exit ($\unicode[STIX]{x1D6FF}_{o}$) exhibits self-similarity in the core region of the spray, but not in the outer zone. In addition, we show that global drop size spectra in the sprays show two distinct characteristics. The spectra from varying $Re$ and $We$ collapse onto a universal p.d.f. for drops of size $x$ where $x/\unicode[STIX]{x1D6FF}_{o}>1$. For $x/\unicode[STIX]{x1D6FF}_{o}<1$, a residual effect of $Re$ and $We$ persists in the size spectra. We explain this characteristic by the fact that the physical mechanisms that cause large drops is different from that which is responsible for the small drops. Similarly, with the liquid sheet velocity at the nozzle exit ($u_{s}$) as the choice of velocity scale, we show that drops moving with a velocity $u$ such that $u/u_{s}<1$ collapse onto a universal p.d.f., while drops with $u/u_{s}>1$ exhibit a residual effect of $Re$ and $We$. From these observations, we suggest that physically accurate models for drop size and velocity spectra should rely on piecewise descriptions of the p.d.f. rather than invoking a single mathematical form for the entire distribution. Finally, we show from a dynamical modal analysis that the conical liquid sheet flapping characteristics exhibit a sharp transition in Strouhal number ($St$) at a critical $Re$.

In this work, we demonstrate the quantification of creatinine in human urine samples using a microcalorimetric sensing system. The calorimetric sensor is based on an array of microfabricated Y-cut quartz resonators. The piezoelectric quartz is etched down to a thickness of 10 μm and exhibits a bulk acoustic resonance of 166 MHz. The temperature sensitivity of this high-frequency quartz resonator is 14 600 Hz/K due to the high phenomenological sensitivity of quartz. Most importantly, the quartz sensors and the analyte fluidics are decoupled providing a significantly more robust calorimetric sensing system than directly contacted chip calorimeters. A reference resonator, consisting of a suspended structure held by four arms, was realized to thermal isolation from the bulk quartz by using focused ion beam etching. We employ alginate entrapped creatinine deiminase to transduce urinary creatinine into temperature signatures, permitting the quantification of creatinine. Fairly good agreement with the measured creatinine values in the 5 urine samples using calorimetric and HPLC methods is obtained.

Self-healing materials, which enable an autonomous repair response to damage, are highly desirable for the long-term reliability of woven or nonwoven textiles. Polyelectrolyte layer-by-layer (LbL) films are of considerable interest as self-healing coatings due to the mobility of the components comprising the film. In this work mechanically stable self-healing films were fabricated through construction of a polyelectrolyte LbL film containing squid ring teeth (SRT) proteins. SRTs are structural proteins with unique self-healing properties and high elastic modulus in both dry and wet conditions (>2 GPa) due to their semicrystalline architecture. We demonstrate LbL construction of multilayers containing native and recombinant SRT proteins capable of self-healing defects. Additionally, we show these films are capable of utilizing functional biomolecules by incorporating an enzyme into the SRT multilayer. Urease was chosen as a model enzyme of interest to test its activity via fluorescence assay. Successful construction of the SRT films demonstrates the use of mechanically stable self-healing coatings, which can incorporate biomolecules for more complex protective functionalities for advanced functional fabrics.

Cellulose microfibrils are pseudocrystalline arrays of cellulose chains that are synthesized by cellulose synthases. The enzymes are organized into large membrane-embedded complexes in which each enzyme likely synthesizes and secretes a β-(1→4) glucan. The relationship between the organization of the enzymes in these complexes and cellulose crystallization has not been explored. To better understand this relationship, we used atomic force microscopy to visualize cellulose microfibril formation from nickel-film-immobilized bacterial cellulose synthase enzymes (BcsA-Bs), which in standard solution only form amorphous cellulose from monomeric BcsA-B complexes. Fourier transform infrared spectroscopy and X-ray diffraction techniques show that surface-tethered BcsA-Bs synthesize highly crystalline cellulose II in the presence of UDP-Glc, the allosteric activator cyclic-di-GMP, as well as magnesium. The cellulose II cross section/diameter and the crystal size and crystallinity depend on the surface density of tethered enzymes as well as the overall concentration of substrates. Our results provide the correlation between cellulose microfibril formation and the spatial organization of cellulose synthases.

In this work, we demonstrate the quantification of creatinine in human urine samples using a micro-calorimetric sensing system. The calorimetric sensor is based on an array of microfabricated Y-cut quartz resonators. The piezoelectric quartz is etched down to a thickness of 10 μm and exhibits a bulk acoustic resonance of 166 MHz. The temperature sensitivity of this high-frequency quartz resonator is 14,600 Hz/K due to the high phenomenological sensitivity of quartz. The sensing resonator structure is thermally isolated from the bulk quartz by fabricating a plate-like cantilevered structure from the etched quartz using focused ion beam etching. Importantly, the quartz sensors and fluidics are decoupled providing a significantly more robust calorimetric sensing system than directly contacted thermopile based chip calorimeters. We employ alginate entrapped creatinine deiminase to transduce urinary creatinine into temperature signatures allowing for the quantification. Ultimately, we envision the development of a handheld calorimetric device similar to that of the glucose meter but with the capability of detecting a much wider range of biomolecules due to the near universality of calorimetric sensing.

The design and development of a calorimetric biosensing system enabling relatively high throughput sample analysis are reported. The calorimetric biosensor system consists of a thin (∼20 μm) micromachined Y-cut quartz crystal resonator (QCR) as a temperature sensor placed in close proximity to a fluidic chamber packed with an immobilized enzyme. Layer by layer enzyme immobilization of urease is demonstrated and its activity as a function of the number of layers, pH, and time has been evaluated. This configuration enables a sensing system where a transducer element is physically separated from the analyte solution of interest and is thereby free from fouling effects typically associated with biochemical reactions occuring on the sensor surface. The performance of this biosensing system is demonstrated by detection of 1-200 mM urea in phosphate buffer via a flow injection analysis (FIA) technique. Miniaturized fluidic systems were used to provide continuous flow through a reaction column. Under this configuration the biosensor has an ultimate resolution of less than 1 mM urea and showed a linear response between 0-50 mM. This work demonstrates a sensing modality in which the sensor itself is not fouled or contaminated by the solution of interest and the enzyme immobilized Kapton® fluidic reaction column can be used as a disposable cartridge. Such a system enables reuse and reliability for long term sampling measurements. Based on this concept a biosensing system is envisioned which can perform rapid measurements to detect biomarkers such as glucose, creatinine, cholesterol, urea and lactate in urine and blood continuously over extended periods of time.

In this paper we report a calorimetrie sensing system for the detection of clinically relevant biomolecules. The calorimetrie biosensor consists of a thin (~20 μm) micromachined Y-cut quartz crystal resonator (QCR) placed in close proximity to a fluidic chamber packed with an immobilized enzyme in alginate beads. This configuration enables a detection mechanism physically separated from the solution of interest and is thereby free from fouling. The performance of this biosensor is demonstrated by detection of urea, creatinine and hydrogen peroxide in phosphate buffer via a flow injection analysis (FIA) technique. This work demonstrates a sensing modality in which the sensor itself is not fouled or contaminated by the solution of interest. Such a system enables reuse and reliability for long term sampling measurements. Further, the immobilized enzyme is easily discarded and replaced with a new reaction column. We envision a biosensing system which can perform rapid measurements to detect biomarkers such as glucose, creatinine, urea and lactate in urine and blood continuously over extended periods of time.

This work focuses on the development and experimental evaluation of micromachined chromatographic columns for use in a commercial gas chromatography (GC) system. A vespel/graphite ferrule based compression sealing technique is presented using which leak-proof fluidic interconnection between the inlet tubing and the microchannel was achieved. This sealing technique enabled separation at temperatures up to 350°C on a μGC column. This paper reports the first high-temperature separations in microfabricated chromatographic columns at these temperatures. A 2m microfabricated column using a double Archimedean spiral design with a square cross-section of 100μm×100μm has been developed using silicon microfabrication techniques. The microfabricated column was benchmarked against a 2m 100μm diameter commercial column and the performance between the two columns was evaluated in tests performed under identical conditions. High temperature separations of simulated distillation (ASTM2887) and polycyclic aromatic hydrocarbons (EPA8310) were performed using the μGC column in temperature programmed mode. The demonstrated μGC column along with the high temperature fixture offers one more solution toward potentially realizing a portable μGC device for the detection of semi-volatile environmental pollutants and explosives without the thermal limitations reported to date with μGC columns using epoxy based interconnect technology.

In this paper we report on the recently demonstrated, quartz resonator based thermal biosensor configured as a biomedical instrumentation system for continuous monitoring of kidney function based on the measurement of urinary creatinine excretion. The biosensor consists of a reaction chamber which is physically separated but located in close proximity to a micromachined quartz resonator-based temperature sensor. The highly effective coupling of heat from the reaction chamber to the quartz resonator due to close proximity (10-50 microns) and the extremely high absorption coefficient of quartz in the 8-12 μm wavelength range renders this system into a very sensitive thermal sensor design. The non-contact measurement results in no confounding mass loading effects on the quartz resonator and therefore provides clear calorimetric data. The sensor uses creatinine iminohydrolase enzyme immobilized on polystyrene films designed for disposable use. We have successfully integrated the sensor with a programmable miniaturized fluidic system from LabSmith® Inc. to form a miniaturized test system. In this paper we present the latest results on noncontact thermal sensor configuration integrated with miniature automated fluidic system that is capable of continuous monitoring of kidney function based on the measurement of urinary creatinine excretion.

In this work, we have developed gas chromatography columns using microfabrication techniques and interfaced them using compression sealing technique to enable high temperature (~400°C) operation. Specifically, we present graphite compression sealing technique to achieve leak-proof fluidic interconnection between the inlet tubing and the microchannel, thus removing the limiting factor of epoxy and allowing for high temperature operations. This approach also represents a significant improvement from previous designs in which the tubing are interfaced to the microfluidic channel from the side of the chip resulting which results in significant band broadening and tailing, as well as making the device less amenable to automatic installation. The microfabricated gas chromatography (μGC) column has been successfully used to perform high temperature gas separation.