Advancement of analytical modes in a multichannel, microfluidic droplet-based sample chopper employing phase-locked detection

Abstract

In this work, we expand upon our recently developed droplet-based sample chopping concepts by introducing a multiplexed fluidic micro-chopper device (μChopper). Six aqueous input channels were integrated with a single oil input, and each of these seven channels was controlled by a pneumatic valve for automated sampling through software control. This improved design, while maintaining high precision in valve-based droplet generation at bandwidths of 0.03 to 0.05 Hz, enabled a variety of analytical modes to be employed on-chip compared to previous devices limited to sample/reference alternations. The device was analytically validated for real-time, continuous calibration with a single sample and five standards; multiplexed analysis during calibration using a mixed mode; and standard addition through spiking of six sample droplets with varying amounts of standard. Finally, the standard addition mode was applied to protein quantification in human serum samples using on-chip, homogeneous fluorescence immunoassays. Ultimately, with only ~1.2 μL of total analyzed solution volume— representing 100-fold and 75-fold reductions in reagent and serum volumes, respectively—we were able to generate full, six-point standard addition curves in only 1.5 min, and results correlated well with those from standard plate-reader equipment. This work thus exploited microfluidic valves for both their automation and droplet phase-locking capabilities, resulting in a micro-analytical tool capable of complex analytical interrogation modes on sub-microliter sample volumes while also leveraging drastic noise rejection via lock-in detection. The multichannel μChopper device should prove particularly useful in analyzing precious biological samples or for dynamic analyses at small volume scales.

Automated microfluidic droplet sampling with integrated, mix-and-read immunoassays to resolve endocrine tissue secretion dynamics

“…Since both pumping patterns were found to dispense equal volumes in each cycle, the 5-step pattern was used in all experiments because it required less time per cycle, thereby increasing the effective flow rate. Compared to more passively controlled or off-chip driven microfluidic systems—using syringe pumps, electroosmosis, pressure, or vacuum that all rely on laminar flow to control the ratio of mixed components—these peristaltic pumps have been validated for highly precise and automated control 26, 31, 39 over the volume metered per pump cycle (Figure S-3). The valves also did not suffer from low frequency fluctuations in metering, and the pulsed flow nature of pumping facilitated mixing of the two components from both side of the Y-channel through chaotic advection.…”

“…After pumping aqueous solution, the gate valve closed and created a new oil-water interface formation. Compared with the passive T-junction droplet formation mechanism, which can suffer from droplet size variations caused by pressure and flow rate differences, the valve segmentation-based droplet formation method used herein has been proven by our group and others 26, 31, 39 to provide accurate and precise volumetric control of droplets.…”

“…Thus, all the samples were premixed and incubated before loading onto the μ Chopper. However, an important feature of the μ Chopper is the ability discriminate very small differences in optical signals (fluorescence 25, 26 , absorbance 32 ) even with high background or at very low concentrations due the drift correction and 1 /f noise reduction. This concept was used herein and was very important for enabling insulin secretion quantification from single islets.…”

“…Based on this concept, we were able to indirectly study insulin secretion dynamics by measuring Zn 2+ (cosecreted with insulin) from single islets at about one-second temporal resolution 24 , although the lack of automation significantly limited adjustments available to this system. Pushing such devices to their sampling limit should theoretically permit sub-second resolution, and automated operation has been shown to offer compelling analytical enhancements 25, 26 .…”

“…Previously, our group developed a lock-in droplet detection system 32 , which modulated a reference signal at the same frequency and phase as the sample by alternatively making sample and reference droplets. Highly, sensitive absorbance 32 and fluorescence 25, 26 detection were achieved with this phase-locked droplet system (μChopper), even at very short optical path lengths (tens of micrometres). Unfortunately, for homogeneous fluorescence immunoassays to be quantified with this lock-in system, a limitation of our previous design is that detection immediately followed droplet formation, which does not allow for the incubation requirement of the immunoassays.…”