Toward a living soft microrobot through optogenetic locomotion control of Caenorhabditis elegans.

Learning from the locomotion of natural organisms is one of the most effective strategies for designing microrobots. However, the development of bioinspired microrobots is still challenging because of technical bottlenecks such as design and seamless integration of high-performance actuation mechanism and high-density energy source for untethered locomotion. Directly harnessing the activation energy and intelligence of living tissues in synthetic micromachines provides an alternative route to developing biohybrid microrobots. Here, we propose an approach to engineering the genetic and nervous systems of a nematode worm, Caenorhabditis elegans, and creating an untethered, highly controllable living soft microrobot (called "RoboWorm"). A living worm is engineered through optogenetic and biochemical methods to shut down the signal transmissions between its neuronal and muscular systems while its muscle cells still remain optically excitable. Through dynamic modeling and experimental verification of the worm crawling, we found that the phase difference between the worm body curvature and the muscular activation pattern generates the thrust force for crawling locomotion. By reproducing the phase difference via optogenetic excitation of the worm body muscles, we emulated the major worm crawling behaviors in a controllable manner. Furthermore, with real-time visual feedback of the worm crawling, we realized closed-loop regulation of the movement direction and destination of single worms. This technology may facilitate scientific studies on the biophysics and neural basis of crawling locomotion of C. elegans and other nematode species.

NanoPADs and nanoFACEs: an optically transparent nanopaper-based device for biomedical applications.

Paper has been a popular material of choice for biomedical applications including for bioanalysis and cell biology studies. Regular cellulose paper-based devices, however, have several key limitations including slow fluid flow; large sample retention in the paper matrix for microfluidic paper-based analytical device (µPAD) application; serious solvent evaporation issues, and contamination and poor control of experimental conditions for cell culture. Here, we describe the development of two novel platforms, nanopaper-based analytical devices (nanoPADs) and nanofibrillated adherent cell-culture platforms (nanoFACEs), that use nanofibrillated cellulose (NFC) paper, simply called nanopaper, as the substrate material to create transparent, pump-free and hollow-channel paper-based microfluidic devices. Due to the natural hydrophilicity and nanoscale pore size of nanopaper, the hollow-channel microfluidic devices can realize a totally pump-free flow without any complicated surface chemical functionalization on the nanopaper. Experimental results showed that within a certain range, larger hollow channel size leads to faster pump-free flows. Different from previous designs of paper-based hollow-channel microfluidic devices, the high transparency of the nanopaper substrate enabled the integration of various optical sensing and imaging technologies together with the nanoPADs and nanoFACEs. As proof-of-concept demonstrations, we demonstrated the use of nanoPADs for colorimetric sensing of glucose and surface-enhanced Raman spectroscopy (SERS)-based detection of environmental pollutants and applied the nanoFACEs to the culture of human umbilical vein endothelial cells (HUVECs). These demonstrations show the great promise of nanoPADs and nanoFACEs for biomedical applications such as chemical/bioanalysis and cell biology studies.

An Automated Microfluidic System for Morphological Measurement and Size-Based Sorting of C. Elegans.

This paper reports a vision-based automated microfluidic system for morphological measurement and size-based sorting of the nematode worm C. elegans. Exceeding the capabilities of conventional worm sorting microfluidic devices purely relying on passive sorting mechanisms, our system is capable of accurate measurement of the worm length/width and active sorting of worms with the desired sizes from a mixture of worms with different body sizes. This function is realized based on the combination of real-time, vision-based worm detection and sizing algorithms and automated on-chip worm manipulation. A double-layer microfluidic device with computer-controlled pneumatic valves is developed for sequential loading, trapping, vision-based sizing, and sorting of single worms. To keep the system operation robust, vision-based algorithms on detecting multi-worm loading and worm sizing failure have also been developed. We conducted sorting experiments on 319 worms and achieved an average sorting speed of 10.4 worms per minute (5.8 s/worm) with an operation success rate of 90.3%. This system will facilitate the worm biology studies where body size measurement and size-based sorting of many worms are needed.

Switched Fuzzy-PD Control of Contact Forces in Robotic Microbiomanipulation.

Force sensing and control are of paramount importance in robotic micromanipulation. A contact force regulator capable of accurately applying mechanical stimuli to a live Drosophila larva could greatly facilitate mechanobiology research on Drosophila and may eventually lead to novel discoveries in mechanotransduction mechanisms of neuronal circuitries. In this paper, we present a novel contact force control scheme implemented in an automated Drosophila larvae micromanipulation system, featuring a switched fuzzy to proportional-differential (PD) controller and a noise-insensitive extended high gain observer (EHGO). The switched fuzzy-PD control law inherits the fast convergence of fuzzy control and overcomes its drawbacks such as large overshoot and steady-state oscillation. The noise-insensitive EHGO can reliably estimate system modeling errors and is robust to force measurement noises, which is advantageous over conventional high gain observers (sensitive to signal noises). Force control experiments show that, compared to a proportional-integral-differential (PID) controller, this new force control scheme significantly enhances the system dynamic performance in terms of rising time, overshoot, and oscillation. The developed robotic system and the force control scheme will be applied to mechanical stimulation and fluorescence imaging of Drosophila larvae for identifying new mechanotransduction mechanisms.

A microfluidic device for automated, high-speed microinjection of Caenorhabditis elegans.

The nematode worm Caenorhabditis elegans has been widely used as a model organism in biological studies because of its short and prolific life cycle, relatively simple body structure, significant genetic overlap with human, and facile/inexpensive cultivation. Microinjection, as an established and versatile tool for delivering liquid substances into cellular/organismal objects, plays an important role in C. elegans research. However, the conventional manual procedure of C. elegans microinjection is labor-intensive and time-consuming and thus hinders large-scale C. elegans studies involving microinjection of a large number of C. elegans on a daily basis. In this paper, we report a novel microfluidic device that enables, for the first time, fully automated, high-speed microinjection of C. elegans. The device is automatically regulated by on-chip pneumatic valves and allows rapid loading, immobilization, injection, and downstream sorting of single C. elegans. For demonstration, we performed microinjection experiments on 200 C. elegans worms and demonstrated an average injection speed of 6.6 worm/min (average worm handling time: 9.45 s/worm) and a success rate of 77.5% (post-sorting success rate: 100%), both much higher than the performance of manual operation (speed: 1 worm/4 min and success rate: 30%). We conducted typical viability tests on the injected C. elegans and confirmed that the automated injection system does not impose significant adverse effect on the physiological condition of the injected C. elegans. We believe that the developed microfluidic device holds great potential to become a useful tool for facilitating high-throughput, large-scale worm biology research.