The content is broken down into the following outline:

Transcription

1 Microfluidics has been around for a long time now, yet there are so many silos of information, and so many folks offering a variety of solutions, it s hard to ask the right questions to ensure you're choosing the right solution for your product concept. This series will focus on integration of an assay from standard laboratory ware, e.g. a microtiter plate, and porting it into a microfluidic cartridge with the assay protocol in the cartridge being managed by an instrument interface, usually a small benchtop or handheld product. To develop a design concept for the overall cartridge, the workflow integration needs to be addressed in a step-wise fashion. Whether the assay protocol is a sandwich immune assays, PCR, and cell-based assays, the same considerations apply in the design development. The objective is to create a set of cartridge specifications that will enable design for manufacture while delivering on the required limit of detection, sensitivity within a limit of variability that ensures useful results are consistently provided. The development process for microfluidics products is especially complex because the biological components have been optimized to perform in standard lab ware, and will need to be re-optimized for a microfluidic device. Part of the challenge is to organize the development in a logical sequence, focusing on resolving big risk areas first, and part of it is looking at the problem from the desired end result - what do you want this system to do and with what level of accuracy and precision? What's good enough vs. what exactly mimics what is currently done in the lab with high quality analytical instrumentation? To help folks who are simply interested in becoming users of microfluidics, yet are in faced with developing a protocol for a microfluidic cartridge, a four part series is presented here that captures the things one should consider when integrating an assay into a microfluidic device. The content is broken down into the following outline: 1) Start at the end. 2) Think modularly. 3) How will I know if I'm getting good results? 4) The Integration Challenge Start at the End From a product perspective, It's all about having the end in mind, and breaking the problem down step by step and working backwards to the beginning. In the case of microfluidics, the beginning is a set of reagents that form an assay protocol or workflow that will be acted on by a set of fluidic components. At the end of the protocol, the result will be measured by a sensor or detector, either optical or electroactive. The objective is to be able to measure things reproducibly, and the microfluidic device is only there to support making repeatable measurements. So the first critical problem to solve is the detection piece. Read More

2 How to Design a Microfluidic Device Part 1: Start at the End Leanna M. Levine, Ph.D., Founder, ALine, Inc. 28 August 2017 For many life science applications, you will likely start by using the same detection platform that you used in the non-microfluidic system to develop the microfluidic device. This could include using a microscope stage, a microtiter plate reader, or even a standard single sample spectrometer/fluorimeter. For these applications, getting the specifications for consumables that are currently used in the system is the best place to start. You can often get these dimensions from the manufacturer or measure it yourself. At this point, all microfluidics suppliers are familiar with the standard footprints used in the life science lab: the microtiter plate, the microscope slide, and a credit card (which is sometimes closer to two microscope slides side by side). In each of these cases, we are talking about some kind of optical measurement, typically fluorescence, but sometimes transmission. In standard instrumentation, especially microscopes, there is also a constraint on the z-height of the device in the detection region, especially for microscopebased measurements. Let s consider a first case in which we are doing a sandwich immunoassay in a microfluidic device. The DETECTION FLOWCELL SPECIFICATION schematic shown above illustrates a typical block MATERIALS diagram for a sandwich assay. The very first thing to FLUORESCENCE: OPTICAL QUALITY MATERIALS address is the spotting of the capture antibody. This CYCLIC OLEFINS, PMMA, OR POLYCARBONATE you can do in a static system spotting the same ELECTROACTIVE: PETG, PMMA, POLYCARBONATE materials you plan to use in the detection chamber DIMENSIONS for the microfluidic. In the bottom of the detection CONSISTENT WITH DETECTION AREA: TYPICALLY area is the capture antibody, and the fluid stream A CHANNEL THAT IS AT LEAST.250 MM WIDER contains the analyte and/or secondary read AND LONGER THAN DETECTION AREA. antibody. So the first set of specifications you can HEIGHT:.025 TO.500 MM. EXTRA LENGTH NEEDED FOR INLET AND OUTLET PORTS. develop are for the detection area in the device. The DISTANCE BETWEEN FEATURES >.75 MM materials, and dimensions in the x-y plane should match the manufacturer s specifications for the detection instrumentation. In the z-direction, the

3 microfluidic detection component will typically involve measuring something that was done statically in an open well, into a system that is measured with flow, in a flow cell. The big changes from a measurement perspective are that the surface area to volume is much higher in the flow cell, and the binding kinetics in a flow vs diffusion in a static system, shortens the time it takes to reach maximum signal. Essentially, you ve taken the volume of fluid that was in a well, and spread it out over the area of the flat flow cell. You ve also added ports and tubing, and thus increased the dead volume in the system. It seems counter intuitive that you would even want to do this, but the flow cell does greatly enhance binding kinetics, especially if you move the fluid through the flow cell to reach the endpoint. Ah ha, you say, don t we want to recirculate the volume then? Yes, you could do that, but you could also make it simpler and adjust the Some Handy Dimensional References: 1 mm x 1mm x 1mm = 1 ul.040 = 1 mm.001 = 1 mil = 25 microns detection limit. flow rate to be slow enough to replenish the concentration at the diffusion boundary layer, increase the concentration of the secondary antibody a little, or do both. For the analyte, you do want to optimize the channel height, the flow rate and the reagent composition to ensure you can reach the required The next issue to address is how you are going to add reagents and control the flow. For general immunoassay protocols, we prefer to start by doing a quick fluid exchange followed by incubation. This can be implemented with a pipettor in the initial stage, followed by a fluid distribution manifold. The fluid distribution manifold allows low dead volume fluid connections to a set of reservoirs. Flow is achieved using on-board pneumatically controlled pumps that access a series of reservoirs attached to the device. A syringe pump requires additional considerations for dead volume in the tubing, and a rotary valve or similar switching method to add different reagents. With a set-up that provides flexibility in flowrates and the ability to move the fluid back and forth, a set of experiments should be designed to explore the optimal geometry of the flowcell, the flow rates, and reagent concentrations and composition, to achieve the maximum signal (with the chosen detection system and capture antibody attachment method). The results collected during this phase can be used to benchmark results to the static system or the gold standard assay as performed in an analytical laboratory environment. An important result will be the specifications for the detection module in the system. Not only is the detection geometry optimized, but new optimal concentrations and compositions for the reagents, materials, as well as the flow rates and wash volumes needed for the upstream fluidic control components is understood. These new specifications will guide integration of other fluid control components into the device. Even early on, a systems approach can be applied to maximize what is learned through optimizing each sub component of the system.

4 For an electroactive detection system, the constraints on the materials is lessened, and integration of the sensor, whether it s a silicon wafer, an electrode on glass, a PCB, or a flex circuit is readily accomplished with room temperature bonding using a pressure sensitive adhesive. In the case of the development of an ion-selective electrode, in which it is required to measure microv changes in the potential, a fluidic test system that is operated with instrument control with the sensor and electrometer in a shielded box, greatly speeds the optimization process. Shown below is an example of a measurement system used to optimize an array of ion-selective electrodes. A video showing the operation of the test cartridge is available here. The pneumatic controller air lines, shown above as a combination of red and blue tubing, provides +/- ~10 psi air in each channel. The tubing is connected along the back end of the sensor test card using flexible tubing and hose barb connections in the fluidic test card. The test card is loaded with three different concentrations of NaCl in order to measure the response of the ion selective electrode formulation spotted on the Ag/AgCl sensors. Once loaded with a pipettor, the card is inserted into the electrometer, where the pre-programmed actuation routine runs the protocol for the measurements. The advantage is that these noise-sensitive measurements can be made in a stable, shielded unit without noise entering the system from the experimentalist. Sample data is shown on the right to illustrate the three different measurements that can be made with three concentrations of NaCl. Using such a test system, the detection chamber geometry, and the fluid actuation routine can be optimized so that, in this case the ISE formulation and the reference electrode can be optimized to achieve the required detection sensitivity. These development activities can be organized into the well-known agile approach used for DETECTION SYSTEM SPECIFICATIONS FLOWCELL GEOMETRY & MATERIALS REAGENTS & COMPOSITION FLOWRATES & VOLUMES DETECTOR CONFIGURATION & COMPONENTS

5 software product development, in which each scrum addresses a needed specification or function of the system. The scrum is divided into sprints in which cross-functional teams work to design and execute the methods and procedures used to create, in this case, the detection portion of the system, including; the geometry of the detection chamber, the reagent composition, and the flowrate, which will meet the detection specifications (lower detection limit, variance, sensitivity and dynamic range). These results can be reproduced and benchmarked as further integration occurs. The learning from this activity is integrated into the next phase of the engineering effort; the step-wise integration of the upstream fluidic components, and allows all successive efforts to be benchmarked to the results generated from the detection module specification. About ALine: Founded on the need for high quality custom microfluidics with a rapid prototyping process that supports a rapid design-build-test cycle, ALine has grown and matured to enable us to support our client s development programs with microfluidics expertise in biology and engineering. Our microfluidics experts had direct experience with integrating assays into microfluidic devices. Contact us at info@alineinc.com or call to discuss your microfluidic requirements. From our beginnings in rapid prototyping 14 years ago, ALine has created a set of engineered fluidhandling components, such as pneumatically controlled on-board valves and pumps, design rules and material sets that work for a variety of assays, including PCR and cell culture. Our development instrumentation is delivered to our customers with the actuation protocol already developed. With our on-site support, we deliver integration of an assay protocol into a microfluidic system in less than four months. This means our clients are busy collecting data and optimizing the assay through a systems integration approach early in the program. Optimization of the assay, microfluidic device design, and the actuation protocol that runs the assay are all developed together in a tight series of design-build-test cycles. The result is a device design and instrument interface that is de-risked for commercialization. Our Team: Dr. Leanna M. Levine, Ph.D. CEO and Founder: Dr. Stefano Begolo, Ph.D. Director of Engineering: Mr. Said Ehrlich, M.S.E.E., Lead Engineer: Mr. Justin Podcerviensky, B.S., Production Manager: Ms. Francesca Yu, B.S., Quality Specialist: llevine@alineinc.com sbegolo@alineinc.com sehrlich@alineinc.com justinp@alineinc.com franyu@alineinc.com