No abstract

The goal of time-resolved cryo-electron microscopy is to determine structural models for transient functional states of large macromolecular complexes such as ribosomes and viruses. The challenge of time-resolved cryo-electron microscopy is to rapidly mix reactants, and then, following a defined time interval, to rapidly deposit them as a thin film and freeze the sample to the vitreous state. Here we describe a methodology in which reaction components are mixed and allowed to react, and are then sprayed onto an EM grid as it is being plunged into cryogen. All steps are accomplished by a monolithic, microfabricated silicon device that incorporates a mixer, reaction channel, and pneumatic sprayer in a single chip. We have found that microdroplets produced by air atomization spread to sufficiently thin films on a millisecond time scale provided that the carbon supporting film is made suitably hydrophilic. The device incorporates two T-mixers flowing into a single channel of four butterfly-shaped mixing elements that ensure effective mixing, followed by a microfluidic reaction channel whose length can be varied to achieve the desired reaction time. The reaction channel is flanked by two ports connected to compressed humidified nitrogen gas (at 50 psi) to generate the spray. The monolithic mixer-sprayer is incorporated into a computer-controlled plunging apparatus. To test the mixing performance and the suitability of the device for preparation of biological macromolecules for cryo-EM, ribosomes and ferritin were mixed in the device and sprayed onto grids. Three-dimensional reconstructions of the ribosomes demonstrated retention of native structure, and 30S and 50S subunits were shown to be capable of reassociation into ribosomes after passage through the device.

Isolating rare cells from biological fluids including whole blood or bone marrow is an interesting biological problem. Characterization of a few metastatic cells from cancer patients for further study is desirable for prognosis/diagnosis. Traditional methods have not proven adequate, due to the compositional complexity of blood, with its large numbers of cell types. To separate individual cells based on their mechanical characteristics, we have developed a series of massively parallel microfabricated sieving device. These devices were constructed with four successively narrower regions of channels numbering approximately 1800 per region. As cells traversed the device, they encountered each region and stopped at a gap width that prohibited passage due to their size. Cultured neuroblastoma cells, when mixed with whole blood and applied to the device, were retained in the 10-microm-wide by 20-microm-deep channels. All other cells migrated to the output. A derivative of the same device was utilized to characterize migration of whole blood. Adult white blood cells were retained at the 2.5-microm-wide by 5-microm-deep channels, while red blood cells passed through these channels. Devices designed to capture rare cells in peripheral circulation for downstream analysis will provide an important tool for diagnosis and treatment.

No abstract

We report the investigation of a novel microfluidic mixing device to achieve submillisecond mixing. The micromixer combines two fluid streams of several microliters per second into a mixing compartment integrated with two T-type premixers and 4 butterfly-shaped in-channel mixing elements. We have employed three dimensional fluidic simulations to evaluate the mixing efficiency, and have constructed physical devices utilizing conventional microfabrication techniques. The simulation indicated thorough mixing at flow rate as low as 6 µL/s. The corresponding mean residence time is 0.44 ms for 90% of the particles simulated, or 0.49 ms for 95% of the particles simulated, respectively. The mixing efficiency of the physical device was also evaluated using fluorescein dye solutions and FluoSphere-red nanoparticles suspensions. The constructed micromixers achieved thorough mixing at the same flow rate of 6 µL/s, with the mixing indices of 96% ± 1%, and 98% ± 1% for the dye and the nanoparticle, respectively. The experimental results are consistent with the simulation data. The device demonstrated promising capabilities for time resolved studies for macromolecular dynamics of biological macromolecules.

Online education is important in the COVID-19 pandemic, but online exam at individual homes invites students to cheat in various ways, especially collusion. While physical proctoring is impossible during social distancing, online proctoring is costly, compromises privacy, and can lead to prevailing collusion. Here we develop an optimization-based anti-collusion approach for distanced online testing (DOT) by minimizing the collusion gain, which can be coupled with other techniques for cheating prevention. With prior knowledge of student competences, our DOT technology optimizes sequences of questions and assigns them to students in synchronized time slots, reducing the collusion gain by 2–3 orders of magnitude relative to the conventional exam in which students receive their common questions simultaneously. Our DOT theory allows control of the collusion gain to a sufficiently low level. Our recent final exam in the DOT format has been successful, as evidenced by statistical tests and a post-exam survey.

This work explores the potential of microwave heating for applications requiring parallel DNA amplification platforms. Device characterization and thermal modeling is performed on 4.1μl microfluidic chamber fabricated in polycarbonate. Microwave power at 6GHz is delivered to the chamber via copper transmission line in a microstrip configuration. Microwave power reflection coefficient and temperature measurements are performed to characterize the power coupled to the chamber and rate of change in temperature. Temperatures up to 72°C are achieved with less than 400mW power applied at the input of the transmission line. Initial heating and cooling rates measured experimentally are ∼7 and ∼6°C∕s, respectively. These results suggest that microwave heating is an efficient, rapid heating technique suitable for programmable, parallel DNA amplification platforms to be empolyed in future genetic analysis systems.

No abstract