High-throughput optofluidic screening for improved microbial cell factories via real-time micron-scale productivity monitoring.

The industrial synthetic biology sector has made huge investments to achieve relevant miniaturized screening systems for scalable fermentation. Here we present the first example of a high-throughput (>103 genotypes per week) perfusion-based screening system to improve small-molecule secretion from microbial strains. Using the Berkeley Lights Beacon® system, the productivity of each strain could be directly monitored in real time during continuous culture, yielding phenotypes that correlated strongly (r2 > 0.8, p < 0.0005) with behavior in industrially relevant bioreactor processes. This method allows a much closer approximation of a typical fed-batch fermentation than conventional batch-like droplet or microplate culture models, in addition to rich time-dependent data on growth and productivity. We demonstrate these advantages by application to the improvement of high-productivity strains using whole-genome random mutagenesis, yielding mutants with substantially improved (by up to 85%) peak specific productivities in bioreactors. Each screen of ∼5 × 103 mutants could be completed in under 8 days (including 5 days involving user intervention), saving ∼50-75% of the time required for conventional microplate-based screening methods.

Determination of equilibrium dissociation constants for recombinant antibodies by high-throughput affinity electrophoresis.

High-quality immunoreagents enhance the performance and reproducibility of immunoassays and, in turn, the quality of both biological and clinical measurements. High quality recombinant immunoreagents are generated using antibody-phage display. One metric of antibody quality - the binding affinity - is quantified through the dissociation constant (KD) of each recombinant antibody and the target antigen. To characterize the KD of recombinant antibodies and target antigen, we introduce affinity electrophoretic mobility shift assays (EMSAs) in a high-throughput format suitable for small volume samples. A microfluidic card comprised of free-standing polyacrylamide gel (fsPAG) separation lanes supports 384 concurrent EMSAs in 30 s using a single power source. Sample is dispensed onto the microfluidic EMSA card by acoustic droplet ejection (ADE), which reduces EMSA variability compared to sample dispensing using manual or pin tools. The KD for each of a six-member fragment antigen-binding fragment library is reported using ~25-fold less sample mass and ~5-fold less time than conventional heterogeneous assays. Given the form factor and performance of this micro- and mesofluidic workflow, we have developed a sample-sparing, high-throughput, solution-phase alternative for biomolecular affinity characterization.

Technologies That Enable Accurate and Precise Nano- to Milliliter-Scale Liquid Dispensing of Aqueous Reagents Using Acoustic Droplet Ejection.

Acoustic liquid handling uses high-frequency acoustic signals that are focused on the surface of a fluid to eject droplets with high accuracy and precision for various life science applications. Here we present a multiwell source plate, the Echo Qualified Reservoir (ER), which can acoustically transfer over 2.5 mL of fluid per well in 25-nL increments using an Echo 525 liquid handler. We demonstrate two Labcyte technologies-Dynamic Fluid Analysis (DFA) methods and a high-voltage (HV) grid-that are required to maintain accurate and precise fluid transfers from the ER at this volume scale. DFA methods were employed to dynamically assess the energy requirements of the fluid and adjust the acoustic ejection parameters to maintain a constant velocity droplet. Furthermore, we demonstrate that the HV grid enhances droplet velocity and coalescence at the destination plate. These technologies enabled 5-µL per destination well transfers to a 384-well plate, with accuracy and precision values better than 4%. Last, we used the ER and Echo 525 liquid handler to perform a quantitative polymerase chain reaction (qPCR) assay to demonstrate an application that benefits from the flexibility and larger volume capabilities of the ER.

Characterizing asthma from a drop of blood using neutrophil chemotaxis.

Asthma is a chronic inflammatory disorder that affects more than 300 million people worldwide. Asthma management would benefit from additional tools that establish biomarkers to identify phenotypes of asthma. We present a microfluidic solution that discriminates asthma from allergic rhinitis based on a patient's neutrophil chemotactic function. The handheld diagnostic device sorts neutrophils from whole blood within 5 min, and generates a gradient of chemoattractant in the microchannels by placing a lid with chemoattractant onto the base of the device. This technology was used in a clinical setting to assay 34 asthmatic (n = 23) and nonasthmatic, allergic rhinitis (n = 11) patients to establish domains for asthma diagnosis based on neutrophil chemotaxis. We determined that neutrophils from asthmatic patients migrate significantly more slowly toward the chemoattractant compared with nonasthmatic patients (P = 0.002). Analysis of the receiver operator characteristics of the patient data revealed that using a chemotaxis velocity of 1.55 µm/min for asthma yields a diagnostic sensitivity and specificity of 96% and 73%, respectively. This study identifies neutrophil chemotaxis velocity as a potential biomarker for asthma, and we demonstrate a microfluidic technology that was used in a clinical setting to perform these measurements.

The present and future role of microfluidics in biomedical research.

Microfluidics, a technology characterized by the engineered manipulation of fluids at the submillimetre scale, has shown considerable promise for improving diagnostics and biology research. Certain properties of microfluidic technologies, such as rapid sample processing and the precise control of fluids in an assay, have made them attractive candidates to replace traditional experimental approaches. Here we analyse the progress made by lab-on-a-chip microtechnologies in recent years, and discuss the clinical and research areas in which they have made the greatest impact. We also suggest directions that biologists, engineers and clinicians can take to help this technology live up to its potential.

Peptidylarginine deiminase 4 contributes to tumor necrosis factor α-induced inflammatory arthritis.

OBJECTIVE: Peptidylarginine deiminase 4 (PAD4) is a citrullinating enzyme that has multiple associations with inflammation. In rheumatoid arthritis, PAD4 and protein citrullination are increased in inflamed joints, and anti-citrullinated protein antibodies (ACPAs) form against citrullinated antigens are formed. ACPA immune complexes can deposit in the joint and induce the production of tumor necrosis factor α (TNFα), a critical inflammatory cytokine in the pathogenesis of rheumatoid arthritis. Further, in other settings, TNFα has been shown to induce PAD4 activity and modulate antibody formation. We undertook this study to investigate whether TNFα and PAD4 may synergistically exacerbate autoantibody production and inflammatory arthritis. METHODS: To determine whether TNFα and PAD4 augment autoantibody production and inflammatory arthritis, we first used a multiplex assay to determine whether mice with chronic inflammatory arthritis due to overexpression of TNFα develop autoantibodies against native and citrullinated antigens. With TNF(+) PAD4(+/+) and TNF(+) PAD4(-/-) mice, we then compared serum autoantibody levels by multiplex array, lymphocyte activation by flow cytometry, total serum IgG levels by enzyme-linked immunosorbent assay, arthritis by clinical and histologic scoring, and systemic inflammation using microfluidic devices. RESULTS: TNFα-overexpressing mice had increased levels of autoantibodies reactive against native and citrullinated antigens. PAD4(-/-) mice with TNFα-induced arthritis had lower levels of autoantibodies reactive against native and citrullinated antigens, decreased T cell activation and total IgG levels, and reduced inflammation and arthritis compared to PAD4(+/+) TNFα-overexpressing mice. CONCLUSION: PAD4 mediates autoantibody production and inflammatory arthritis downstream of TNFα.

Microfluidic kit-on-a-lid: a versatile platform for neutrophil chemotaxis assays.

Improvements in neutrophil chemotaxis assays have advanced our understanding of the mechanisms of neutrophil recruitment; however, traditional methods limit biologic inquiry in important areas. We report a microfluidic technology that enables neutrophil purification and chemotaxis on-chip within minutes, using nanoliters of whole blood, and only requires a micropipette to operate. The low sample volume requirements and novel lid-based method for initiating the gradient of chemoattractant enabled the measurement of human neutrophil migration on a cell monolayer to probe the adherent and migratory states of neutrophils under inflammatory conditions; mouse neutrophil chemotaxis without sacrificing the animal; and both 2D and 3D neutrophil chemotaxis. First, the neutrophil chemotaxis on endothelial cells revealed 2 distinct neutrophil phenotypes, showing that endothelial cell-neutrophil interactions influence neutrophil chemotactic behavior. Second, we validated the mouse neutrophil chemotaxis assay by comparing the adhesion and chemotaxis of neutrophils from chronically inflamed and wild-type mice; we observed significantly higher neutrophil adhesion in blood obtained from chronically inflamed mice. Third, we show that 2D and 3D neutrophil chemotaxis can be directly compared using our technique. These methods allow for new avenues of research while reducing the complexity, time, and sample volume requirements to perform neutrophil chemotaxis assays.

Rapid prototyping of arrayed microfluidic systems in polystyrene for cell-based assays.

Microfluidic cell-based systems have enabled the study of cellular phenomena with improved spatiotemporal control of the microenvironment and at increased throughput. While poly(dimethylsiloxane) (PDMS) has emerged as the most popular material in microfluidics research, it has specific limitations that prevent microfluidic platforms from achieving their full potential. We present here a complete process, ranging from mold design to embossing and bonding, that describes the fabrication of polystyrene (PS) microfluidic devices with similar cost and time expenditures as PDMS-based devices. Emphasis was placed on creating methods that can compete with PDMS fabrication methods in terms of robustness, complexity, and time requirements. To achieve this goal, several improvements were made to remove critical bottlenecks in existing PS embossing methods. First, traditional lithographic techniques were adapted to fabricate bulk epoxy molds capable of resisting high temperatures and pressures. Second, a method was developed to emboss through-holes in a PS layer, enabling creation of large arrays of independent microfluidic systems on a single device without need to manually create access ports. Third, thermal bonding of PS layers was optimized in order to achieve quality bonding over large arrays of microsystems. The choice of materials and methods was validated for biological function in two different cell-based applications to demonstrate the versatility of our streamlined fabrication process.