Single-molecule force spectroscopy is crucial for understanding molecular biophysics and mechanobiology, but existing techniques like magnetic tweezers, atomic force microscopy (AFM), and optical tweezers suffer from limitations in throughput and cost. These methods often test only one or a few interactions at a time, requiring complex and expensive instrumentation. While recent advancements such as centrifugal force microscopy, acoustic bead manipulation, wide-range magnetic fields, and nanophotonic bead trapping have improved multiplexing and reduced cost per experiment, further improvements are needed to make single-molecule force spectroscopy more accessible. Hydrodynamic force spectroscopy, a simpler and cheaper technique, offers multiplexed mechanical loading but is limited to a single loading rate. The need for a cost-effective, high-throughput, and portable approach with simultaneous multi-force testing capabilities led to the development of the FLO-Chip. This research presents the FLO-Chip, a microfluidic device designed to address these limitations. It allows for massively parallel application of mechanical force using fluid flow, a readily implementable method that eliminates the need for specialized instrumentation. The low cost (~$3 per chip) is achieved through the use of PDMS-based soft lithography. The design of the FLO-Chip incorporates serially connected microchannels of varying widths, enabling simultaneous testing at multiple loading rates and significantly increasing throughput (up to ~4000 measurements per chip in ~2 hours). The simplicity of the setup allows for compatibility with standard microscopy equipment found in various biology, biophysics, biomaterials, and bioengineering laboratories, removing the need for specialized force spectroscopy instrumentation. The high throughput and low cost of this method are intended to make single molecule force spectroscopy more accessible and facilitate new applications in the field.

Existing force spectroscopy methods, while powerful, often pose significant hurdles for many laboratories due to their cost and low throughput. Magnetic tweezers and centrifugal force microscopy offer multiplexing, but are limited in the number of interactions that can be tracked simultaneously in a single field of view. Hydrodynamic force spectroscopy presents a lower-cost alternative but is typically limited to a single loading rate. The development of the FLO-Chip is motivated by the need to improve both the cost-effectiveness and throughput of single-molecule force spectroscopy. The study draws upon and builds on the principles of hydrodynamic force spectroscopy and microfluidic devices to achieve its goals. The work references several previous studies that have made contributions to the field of multiplexed force spectroscopy and hydrodynamic force spectroscopy, highlighting both their advancements and limitations. Specific examples cited include research involving centrifugal force microscopy, acoustic bead manipulation, wide-range magnetic fields, nanophotonic bead trapping and various approaches to hydrodynamic force spectroscopy. The review of existing techniques provides the rationale for the FLO-Chip's design and highlights the innovative aspects of the new approach.

The FLO-Chip consists of serially connected microchannels with varying widths (e.g., 500 µm, 1000 µm, 1500 µm, 2000 µm, and 2500 µm) and a fixed height (120 µm). The varying channel widths allow for the application of different drag forces on tethered microbeads at a constant flow rate. The microfluidic device is fabricated using SU-8 photolithography and PDMS soft lithography, which is a low-cost and readily accessible method. The coverslip surface is functionalized with polyethylene glycol (PEG) and biotinylated-PEG to provide a surface for streptavidin binding. The molecular constructs of interest are tethered between the coverslip and microbeads, utilizing biotin-streptavidin binding for stable surface anchoring and a 5745 bp double-stranded DNA (dsDNA) tether. The force is applied by subjecting the tethered beads to controlled fluid flow. The drag force on the beads is calibrated using the equipartition theorem, relating the mean-square displacement of the bead center in the transverse direction to the applied force. The force-extension behavior of the dsDNA tether is verified using the worm-like chain (WLC) model. Different molecular interactions (biotin-streptavidin, digoxigenin-antidigoxigenin, and DNA unzipping) are tested by annealing appropriate single-stranded DNA oligos to the tether. The rupture forces of these interactions are measured under varying loading rates and solution conditions. The data are analyzed using the Evans and Ritchie rupture model to extract parameters such as the most probable rupture force (F*) and the distance to the transition state (Δx). For automated high-throughput analysis, a modified FLO-Chip design with channels of varying width in a single field of view is used. Automated bead selection and rupture detection algorithms are implemented using MATLAB to streamline the data analysis, significantly reducing processing time. The study employs statistical methods like random selection without replication to analyze the rupture force distributions and calculates the mean and standard deviation. Custom MATLAB code for subpixel bead tracking and rupture force analysis is detailed, building upon previously published work. The analysis methods are thoroughly described and validated through comparison with results from previous research using different force spectroscopy techniques.

The FLO-Chip successfully demonstrated massively parallel single-molecule force spectroscopy with significantly enhanced throughput compared to existing methods. The study verified the force calibration method by observing the expected worm-like chain behavior of dsDNA tethers under controlled tension. The rupture forces for biotin-streptavidin and digoxigenin-antidigoxigenin interactions were measured under various loading rates and showed good agreement with values reported in previous studies using AFM and acoustic force spectroscopy. The dependence of rupture force on loading rate was successfully modeled using the Evans and Ritchie rupture model. The influence of GC content on the unzipping force of dsDNA was investigated and confirmed the expected increase in rupture force with higher GC content. The study also demonstrated the ability to investigate the impact of solution conditions by observing how Mg²⁺ concentration affected the rupture force of DNA unzipping. The high-throughput version of the FLO-Chip enabled simultaneous testing of four different loading rates on a single chip, analyzing thousands of events with automated bead selection and rupture detection. This version also successfully characterized the rupture forces of digoxigenin-antidigoxigenin interactions and 9nt DNA unzipping. The Evans and Ritchie model was again used to extract kinetic parameters. Finally, the researchers applied the FLO-Chip to examine the effect of mechanical strain on toehold-mediated strand displacement, revealing a dependence of reaction rate on the location of the applied force (strained or strain-free toehold). Modest increases in the reaction rate at low forces were observed when the toehold was under strain. Significantly higher strand displacement rates were observed at higher forces (>5 pN) when only the branch migration domain was under strain.

The findings confirm that FLO-Chip is a robust and reliable method for single-molecule force spectroscopy, achieving similar accuracy to more expensive and complex techniques while dramatically increasing throughput and reducing cost. The agreement between FLO-Chip results and previously published data validates the accuracy and reliability of this novel approach. The high throughput enabled by the FLO-Chip allows for a more comprehensive characterization of molecular interactions by testing a wider range of loading rates and conditions. The ability to simultaneously test multiple loading rates on a single chip drastically reduces experimental time and resources. The automation of bead selection and rupture detection further enhances the efficiency of the method. The application of FLO-Chip to studying toehold-mediated strand displacement demonstrates its utility in studying complex molecular mechanisms under various mechanical loads, revealing the influence of force on different stages of the process. This study's results have implications for various fields, including biophysics, mechanobiology, and nanotechnology. The low cost and accessibility of FLO-Chip opens new avenues for research by enabling researchers with limited resources to perform high-quality single-molecule force spectroscopy experiments.

This research successfully introduced the FLO-Chip, a low-cost, high-throughput microfluidic platform for massively parallel single-molecule force spectroscopy. The validation experiments demonstrated the accuracy and reliability of the technique, showing strong agreement with previously reported results from other force spectroscopy methods. The high throughput, automation, and low cost of the FLO-Chip make it a powerful tool for studying various molecular interactions, and its application to examining the effects of mechanical force on strand displacement highlights its broad applicability. Future research could focus on further optimizing the chip design, exploring different microchannel geometries, and expanding the types of molecular interactions that can be studied. Integrating the FLO-Chip with advanced microscopy techniques could further enhance its throughput and capabilities. The potential applications extend to various research and educational settings, as well as point-of-care diagnostics.

While the FLO-Chip offers significant advantages, several limitations should be noted. The current design's force calibration is specific to the bead size, tether length, and microchannel height used in this study; recalibration is required if these parameters are altered. The automated analysis relies on image processing algorithms, and the accuracy could be affected by image quality or the presence of noise. The spatial and temporal resolution of the FLO-Chip are limited by the capabilities of the imaging system used, restricting the investigation of extremely rapid or small-scale events. While the throughput is greatly enhanced compared to other methods, there is still a practical limit to the number of molecules that can be tested per chip, especially when utilizing lower-resolution imaging systems. The effect of the elastic DNA tether on the measurement of rupture forces should be carefully considered, particularly for interactions near the coverslip surface.