Robotic agricultural instrument for automated extraction of nematode cysts and eggs from soil to improve integrated pest management.

Soybeans are an important crop for global food security. Every year, soybean yields are reduced by numerous soybean diseases, particularly the soybean cyst nematode (SCN). It is difficult to visually identify the presence of SCN in the field, let alone its population densities or numbers, as there are no obvious aboveground disease symptoms. The only definitive way to assess SCN population densities is to directly extract the SCN cysts from soil and then extract the eggs from cysts and count them. Extraction is typically conducted in commercial soil analysis laboratories and university plant diagnostic clinics and involves repeated steps of sieving, washing, collecting, grinding, and cleaning. Here we present a robotic instrument to reproduce and automate the functions of the conventional methods to extract nematode cysts from soil and subsequently extract eggs from the recovered nematode cysts. We incorporated mechanisms to actuate the stage system, manipulate positions of individual sieves using the gripper, recover cysts and cyst-sized objects from soil suspended in water, and grind the cysts to release their eggs. All system functions are controlled and operated by a touchscreen interface software. The performance of the robotic instrument is evaluated using soil samples infested with SCN from two farms at different locations and results were comparable to the conventional technique. Our new technology brings the benefits of automation to SCN soil diagnostics, a step towards long-term integrated pest management of this serious soybean pest.

Adhesive Tape Microfluidics with an Autofocusing Module That Incorporates CRISPR Interference: Applications to Long-Term Bacterial Antibiotic Studies.

The ability to study bacteria at the single cell level has advanced our insights into microbial physiology and genetics in ways not attainable by studying large populations using more traditional culturing methods. To improve methods to characterize bacteria at the cellular level, we developed a new microfluidic platform that enables cells to be exposed to metabolites in a gradient of concentrations. By designing low-cost, three-dimensional devices with adhesive tapes and tailoring them for bacterial imaging, we avoided the complexities of silicon and polymeric microfabrication. The incorporation of an agarose membrane as the resting substrate, along with a temperature-controlled environmental chamber, allows the culturing of bacterial cells for over 10 h under stable growth or inhibition conditions. Incorporation of an autofocusing module helped the uninterrupted, high-resolution observation of bacteria at the single-cell and at low density population levels. We used the microfluidic platform to record morphological changes in Escherichia coli during ampicillin exposure and to quantify the minimum inhibitory concentration of the antibiotic. We further demonstrated the potential of finely-tuned, incremental gene regulation in a concentration gradient utilizing CRISPR interference (CRISPRi). These low-cost engineering tools, when implemented in combination with genetic approaches such as CRISPRi, should prove useful to uncover new genetic determinants of antibiotic susceptibility and evaluate the long-term effectiveness of antibiotics in bacterial cultures.

New methods of removing debris and high-throughput counting of cyst nematode eggs extracted from field soil.

The soybean cyst nematode (SCN), Heterodera glycines, is the most damaging pathogen of soybeans in the United States. To assess the severity of nematode infestations in the field, SCN egg population densities are determined. Cysts (dead females) of the nematode must be extracted from soil samples and then ground to extract the eggs within. Sucrose centrifugation commonly is used to separate debris from suspensions of extracted nematode eggs. We present a method using OptiPrep as a density gradient medium with improved separation and recovery of extracted eggs compared to the sucrose centrifugation technique. Also, computerized methods were developed to automate the identification and counting of nematode eggs from the processed samples. In one approach, a high-resolution scanner was used to take static images of extracted eggs and debris on filter papers, and a deep learning network was trained to identify and count the eggs among the debris. In the second approach, a lensless imaging setup was developed using off-the-shelf components, and the processed egg samples were passed through a microfluidic flow chip made from double-sided adhesive tape. Holographic videos were recorded of the passing eggs and debris, and the videos were reconstructed and processed by custom software program to obtain egg counts. The performance of the software programs for egg counting was characterized with SCN-infested soil collected from two farms, and the results using these methods were compared with those obtained through manual counting.

A fast, reconfigurable flow switch for paper microfluidics based on selective wetting of folded paper actuator strips.

In paper microfluidics, the development of smart and versatile switches is critical for the regulation of fluid flow across multiple channels. Past approaches in creating switches are limited by long response times, large actuation fluid volumes, and use of external control circuitry. We seek to mitigate these difficulties through the development of a unique actuator device made entirely out of chromatography paper and incorporated with folds. Selective wetting of the fold with an actuation fluid, either at the crest or trough, serves to raise or lower the actuator's tip and thus engage or break the fluidic contact between channels. Here the actuator's response time is dramatically reduced (within two seconds from wetting) and a very small volume of actuation fluid is consumed (four microliters). Using this actuation principle, we implement six switch configurations which can be grouped as single-pole single-throw (normally OFF and normally ON) and single-pole double-throw (with single and double break). By employing six actuators in parallel, an autonomous colorimetric assay is built to detect the presence of three analytes - glucose, protein, and nitrite - in artificial saliva. Finally, this work brings the concept of origami to paper microfluidics where multiple-fold geometries can be exploited for programmable switching of fluidic connections.

Flexible and disposable paper- and plastic-based gel micropads for nematode handling, imaging, and chemical testing.

Today, the area of point-of-care diagnostics is synonymous with paper microfluidics where cheap, disposable, and on-the-spot detection toolkits are being developed for a variety of chemical tests. In this work, we present a novel application of microfluidic paper-based analytical devices (µPADs) to study the behavior of a small model nematode, Caenorhabditis elegans. We describe schemes of µPAD fabrication on paper and plastic substrates where membranes are created in agarose and Pluronic gel. Methods are demonstrated for loading, visualizing, and transferring single and multiple nematodes. Using an anthelmintic drug, levamisole, we show that chemical testing on C. elegans is easily performed because of the open device structure. A custom program is written to automatically recognize individual worms on the µPADs and extract locomotion parameters in real-time. The combination of µPADs and the nematode tracking program provides a relatively low-cost, simple-to-fabricate imaging and screening assay (compared to standard agarose plates or polymeric microfluidic devices) for non-microfluidic, nematode laboratories.