automating-citrus-budwood-processing-for-downstream-pathogen

Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering

Graft-transmissible, phloem-limited pathogens of citrus such as viruses, viroids, and bacteria are responsible for devastating epidemics and serious ...

University of California Riverside

The development of new biological and chemical instruments for research and diagnostic applications is often slowed by the cost, specialization, and custom nature of these instruments. New instruments are built from components that are drawn from a host of different disciplines and not designed to integrate together, and once built, an instrument typically performs a limited number of tasks and cannot be easily adapted for new applications. Consequently, the process of inventing new instruments is very inefficient, especially for researchers or clinicians in resource-limited settings. To improve this situation, we propose that a family of standardized multidisciplinary components is needed, a set of "building blocks" that perform a wide array of different tasks and are designed to integrate together. Using these components, scientists, engineers, and clinicians would be able to build custom instruments for their own unique needs quickly and easily. In this work we present the foundation of this set of components, a system we call Multifluidic Evolutionary Components (MECs). "Multifluidic" conveys the wide range of fluid volumes MECs operate upon (from nanoliters to milliliters and beyond); "multi" also reflects the multiple disciplines supported by the system (not only fluidics but also electronics, optics, and mechanics). "Evolutionary" refers to the design principles that enable the library of MEC parts to easily grow and adapt to new applications. Each MEC "building block" performs a fundamental function that is commonly found in biological or chemical instruments, functions like valving, pumping, mixing, controlling, and sensing. Each MEC also has a unique symbol linked to a physical definition, which enables instruments to be designed rapidly and efficiently using schematics. As a proof-of-concept, we use MECs to build a variety of instruments, including a fluidic routing and mixing system capable of manipulating fluid volumes over five orders of magnitude, an acid-base titration instrument suitable for use in schools, and a bioreactor suitable for maintaining and analyzing cell cultures in research and diagnostic applications. These are the first of many instruments that can be built by researchers, clinicians, and students using the MEC system.

MECs: "Building Blocks" for Creating Biological and Chemical Instruments.

In this work we created functional microfluidic chips without actually designing them. We accomplished this by first generating a library of thousands of different random microfluidic chip designs, then simulating the behavior of each design on a computer using automated finite element analysis. The simulation results were then saved to a database which a user can query via to find chip designs suitable for a specific task. To demonstrate this functionality, we used our library to select chip designs that generate any three desired concentrations of a solute. We also fabricated and tested 16 chips from the library, confirmed that they function as predicted, and used these chips to perform a cell growth rate assay. This is one of many different applications for randomly-designed microfluidics; in principle, any microfluidic chip that can be simulated could be designed automatically using our method. Using this approach, individuals with no training in microfluidics can obtain custom chip designs for their own unique needs in just a few seconds.

Random design of microfluidics.

Microfluidics researchers are increasingly using computer simulation in many different aspects of their research. However, these simulations are often computationally intensive: simulating the behavior of a simple microfluidic chip can take hours to complete on typical computing hardware, and even powerful workstations can lack the computational capabilities needed to simulate more complex chips. This slows the development of new microfluidic chips for new applications. To address this issue, we present a microfluidic simulation method that can simulate the behavior of fluids and particles in some typical microfluidic chips instantaneously (in around one second). Our method decomposes the chip into its primary components: channels and intersections. The behavior of fluid in each channel is determined by leveraging analogies with electronic circuits, and the behavior of fluid and particles in each intersection is determined by querying a database containing nearly 100,000 pre-simulated channel intersections. While constructing this database takes a nontrivial amount of computation time, once built, this database can be queried to determine the behavior of fluids and particles in a given intersection in a fraction of a second. Using this approach, the behavior of a microfluidic chip can be simulated in just one second on a standard laptop computer, without any noticeable degradation in the accuracy of the simulation. While our current technique has some constraints on the designs of the chips it can simulate (namely, T- or cross-shaped intersections, 90 degree channel turns, a fixed channel width, fluid flow rates between 0 and 2 cm/s, and particles with diameters between 1 and 20 microns), we provide several strategies for increasing the range of possible chip designs that can be simulated using our technique. As a proof of concept, we show that our simulation method can instantaneously simulate the paths followed by particles in both simple and complex microfluidic chips, with results that are essentially indistinguishable from simulations that took hours or even days to complete using conventional approaches.

Instantaneous simulation of fluids and particles in complex microfluidic devices.

Measuring the temperature of a sample is a fundamental need in many biological and chemical processes. When the volume of the sample is on the microliter or nanoliter scale (e.g., cells, microorganisms, precious samples, or samples in microfluidic devices), accurate measurement of the sample temperature becomes challenging. In this work, we demonstrate a technique for accurately determining the temperature of microliter volumes using a simple 3D-printed microfluidic chip. We accomplish this by first filling "microfluidic thermometer" channels on the chip with substances with precisely known freezing/melting points. We then use a thermoelectric cooler to create a stable and linear temperature gradient along these channels within a measurement region on the chip. A custom software tool (available as online Supporting Information) is then used to find the locations of solid-liquid interfaces in the thermometer channels; these locations have known temperatures equal to the freezing/melting points of the substances in the channels. The software then uses the locations of these interfaces to calculate the temperature at any desired point within the measurement region. Using this approach, the temperature of any microliter-scale on-chip sample can be measured with an uncertainty of about a quarter of a degree Celsius. As a proof-of-concept, we use this technique to measure the unknown freezing point of a 50 microliter volume of solution and demonstrate its feasibility on a 400 nanoliter sample. Additionally, this technique can be used to measure the temperature of any on-chip sample, not just near-zero-Celsius freezing points. We demonstrate this by using an oil that solidifies near room temperature (coconut oil) in a microfluidic thermometer to measure on-chip temperatures well above zero Celsius. By providing a low-cost and simple way to accurately measure temperatures in small volumes, this technique should find applications in both research and educational laboratories.

A microfluidic thermometer: Precise temperature measurements in microliter- and nanoliter-scale volumes.

Paper microfluidic devices (including lateral flow assays) offer an excellent combination of utility and low cost. Many paper microfluidic devices are fabricated using the Xerox ColorQube line of commercial wax-based color printers; the wax ink serves as a hydrophobic barrier to fluid flow. These printers are capable of depositing four different colors of ink, cyan (C), magenta (M), yellow (Y), and black (K), plus 11 combinations of these colors (CM, CY, CK, MY, MK, YK, CMY, CMK, CYK, MYK, and CMYK), although most researchers use only black ink to print paper microfluidic devices. Recently, as part of a project to develop a computer-aided design framework for use with paper microfluidics devices, we unexpectedly observed that different colors of wax ink behave differently in paper microfluidics. We found that among the single colors of ink, black ink actually had the most barrier failures, and magenta ink had the fewest barrier failures. In addition, some combinations of colors performed even better than magenta: the combinations CY, MK, YK, CMY, CYK and MYK had no barrier failures in our study. We also found that the printer delivers significantly different amounts of ink to the paper for the different color combinations, and in general, the color combinations that formed the strongest barriers to fluid flow were the ones that had the most ink delivered to the paper. This suggests that by simply weighing paper samples printed with all 15 combinations of colors, one can easily find the color combinations most likely to form a strong barrier for a given printer. Finally, to show that deliberate choices of ink colors can actually be used to create new functions in paper microfluidics, we designed and tested a new color-based "antifuse" structure that protects paper microfluidic devices from a typical operator error (addition of too much fluid to the device). Our results provide a set of color choice guidelines that designers can use to control the behavior of their paper microfluidics.

Using printer ink color to control the behavior of paper microfluidics.

The ability to thoroughly mix two fluids is a fundamental need in microfluidics. While a variety of different microfluidic mixers have been designed by researchers, it remains unknown which (if any) of these mixers are optimal (that is, which designs provide the most thorough mixing with the smallest possible fluidic resistance across the mixer). In this work, we automatically designed and rationally optimized a microfluidic mixer. We accomplished this by first generating a library of thousands of different randomly designed mixers, then using the non-dominated sorting genetic algorithm II (NSGA-II) to optimize the random chips in order to achieve Pareto efficiency. Pareto efficiency is a state of allocation of resources (e.g. driving force) from which it is impossible to reallocate so as to make any one individual criterion better off (e.g. pressure drop) without making at least one individual criterion (e.g. mixing performance) worse off. After 200 generations of evolution, Pareto efficiency was achieved and the Pareto-optimal front was found. We examined designs at the Pareto-optimal front and found several design criteria that enhance the mixing performance of a mixer while minimizing its fluidic resistance; these observations provide new criteria on how to design optimal microfluidic mixers. Additionally, we compared the designs from NSGA-II with some popular microfluidic mixer designs from the literature and found that designs from NSGA-II have lower fluidic resistance with similar mixing performance. As a proof of concept, we fabricated three mixer designs from 200 generations of evolution and one conventional popular mixer design and tested the performance of these four mixers. Using this approach, an optimal design of a passive microfluidic mixer is found and the criteria of designing a passive microfluidic mixer are established.

Finding the optimal design of a passive microfluidic mixer.

Many solid-dose oral drug products are engineered to release their active ingredients into the body at a certain rate. Techniques for measuring the dissolution or degradation of a drug product in vitro play a crucial role in predicting how a drug product will perform in vivo. However, existing techniques are often labor-intensive, time-consuming, irreproducible, require specialized analytical equipment, and provide only "snapshots" of drug dissolution every few minutes. These limitations make it difficult for pharmaceutical companies to obtain full dissolution profiles for drug products in a variety of different conditions, as recommended by the US Food and Drug Administration. Additionally, for drug dosage forms containing multiple controlled-release pellets, particles, beads, granules, etc. in a single capsule or tablet, measurements of the dissolution of the entire multi-particle capsule or tablet are incapable of detecting pellet-to-pellet variations in controlled release behavior. In this work, we demonstrate a simple and fully-automated technique for obtaining dissolution profiles from single controlled-release pellets. We accomplished this by inverting the drug dissolution problem: instead of measuring the increase in the concentration of drug compounds in the solution during dissolution (as is commonly done), we monitor the decrease in the buoyant mass of the solid controlled-release pellet as it dissolves. We weigh single controlled-release pellets in fluid using a vibrating tube sensor, a piece of glass tubing bent into a tuning-fork shape and filled with any desired fluid. An electronic circuit keeps the glass tube vibrating at its resonance frequency, which is inversely proportional to the mass of the tube and its contents. When a pellet flows through the tube, the resonance frequency briefly changes by an amount that is inversely proportional to the buoyant mass of the pellet. By passing the pellet back-and-forth through the vibrating tube sensor, we can monitor its mass as it degrades or dissolves, with high temporal resolution (measurements every few seconds) and mass resolution (700 nanogram resolution). As a proof-of-concept, we used this technique to measure the single-pellet dissolution profiles of several commercial controlled-release proton pump inhibitors in simulated stomach and intestinal contents, as well as comparing name-brand and generic formulations of the same drug. In each case, vibrating tube sensor data revealed significantly different dissolution profiles for the different drugs, and in some cases our method also revealed differences between different pellets from the same drug product. By measuring any controlled-release pellets, particles, beads, or granules in any physiologically-relevant environment in a fully-automated fashion, this method can augment and potentially replace current dissolution tests and support product development and quality assurance in the pharmaceutical industry.

Measuring dissolution profiles of single controlled-release drug pellets.

Paper microfluidic or lateral flow devices have found many applications, especially in medical diagnostics. Their low cost and ease of use makes them particularly valuable in resource-limited and point-of-care applications. However, the process of developing new paper microfluidic devices is slowed by the need to find optimal values for their various design parameters, which determine the overall size and fluid volume requirements of the device. Often, researchers must design and test several different versions of a device to find a combination of parameters that functions as intended. To accelerate the development of new paper microfluidics, we developed a software framework that automatically designs custom paper microfluidic devices for a given application. Once the user specifies the desired device parameters, the software generates printable image files of the resulting devices, ready to output to a conventional wax ink color printer and test. As a proof-of-concept, we used our software to automatically design 51 different paper microfluidic devices we needed to create a functional lateral flow assay that detects protein and glucose in urine. These designs took only a few seconds to generate and were used in 120 lab experiments we performed in 16 h in the lab. Thus, with the help of our software framework, we went from an idea to a functional device in just two work days. By accelerating device development and enabling researchers without microfluidics experience to create custom devices, our software can help spread paper microfluidic technology to important new application areas.

Rapid development and optimization of paper microfluidic designs using software automation.

Counterfeit or substandard medicines adversely affect the health of millions of people and cost an estimated $200 billion USD annually. Their burden is greatest in developing countries, where the World Health Organization estimates that one in ten medical products are fake. In this work, I describe a simple addition to the existing drug manufacturing process that imparts an edible universally unique physical identifier to each pill, tablet, capsule, caplet, etc. This technique uses nonpareils (also called sprinkles and "hundreds and thousands"), tiny inexpensive multicolor candy spheres that are normally added to other candies or desserts as decorations. If nonpareils are applied at random to a pill immediately after manufacture, the specific pattern they form is unlikely to ever be repeated by random chance; this means that the pattern (or "CandyCode") can be used to uniquely identify the pill and distinguish it from all other pills. By taking a photograph of each CandyCoded pill after manufacture and recording the location and color of each nonpareil, a manufacturer can construct a database containing the CandyCodes of all known-authentic pills they produce. A consumer can then simply use a cellphone to photograph a pill and transfer its image to the manufacturer's server, which determines whether the pill's CandyCode matches a known-good CandyCode in their database (meaning that the pill is authentic) or does not have a match in the database (in which case the consumer is warned that the pill may be counterfeit and should not be consumed). To demonstrate the feasibility of using random particles as universal identifiers, I performed a series of experiments using both real CandyCodes (on commercially produced chocolate candies) and simulated CandyCodes (generated by software). I also developed a simple method for converting a CandyCode photo to a set of strings for convenient storage and retrieval in a database. Even after subjecting CandyCodes to rough handling to simulate shipping conditions, the CandyCodes were still easily verifiable using a cellphone camera. A manufacturer could produce at least [Formula: see text] CandyCoded pills-41 million for each person on Earth-and still be able to uniquely identify each CandyCode. By providing universally-unique IDs that are easy to manufacture but hard to counterfeit, require no alteration of the existing drug formulation and minimal alteration of the manufacturing process, and need only a cameraphone for verification, CandyCodes could play an important role in the fight against fraud in pharmaceuticals and many other products.

Article	Year
MECs: "Building Blocks" for Creating Biological and Chemical Instruments. PloS one\| PubMed ID: 27437989	2016
Random design of microfluidics. Lab on a chip\| PubMed ID: 27713978	2016
Instantaneous simulation of fluids and particles in complex microfluidic devices. PloS one\| PubMed ID: 29267312	2017
A microfluidic thermometer: Precise temperature measurements in microliter- and nanoliter-scale volumes. PloS one\| PubMed ID: 29284028	2017
Using printer ink color to control the behavior of paper microfluidics. Lab on a chip\| PubMed ID: 31049521	2019
Finding the optimal design of a passive microfluidic mixer. Lab on a chip\| PubMed ID: 31576868	2019
Measuring dissolution profiles of single controlled-release drug pellets. Scientific reports\| PubMed ID: 33184351	2020
Rapid development and optimization of paper microfluidic designs using software automation. Analytica chimica acta\| PubMed ID: 34625247	2021
CandyCodes: simple universally unique edible identifiers for confirming the authenticity of pharmaceuticals. Scientific reports\| PubMed ID: 35523794	2022

William Grover

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