RESUMO
Fluorescence is widely used for small-volume analysis and is a primary tool for on-chip detection in microfluidic devices, yet additional expertise, more elaborate optics, and phase-locked detectors are needed for ultrasensitive measurements. Recently, we designed a microfluidic analog to an optical beam chopper (µChopper) that alternated formation of picoliter volume sample and reference droplets. Without complex optics, the device negated large signal drifts (1/f noise), allowing absorbance detection in a mere 27 µm optical path. Here, we extend the µChopper concept to fluorescence detection with standard wide-field microscope optics. Precision of droplet control in the µChopper was improved by automation with pneumatic valves, allowing fluorescence measurements to be strictly phase locked at 0.04 Hz bandwidth to droplets generated at 3.50 Hz. A detection limit of 12 pM fluorescein was achieved when sampling 20 droplets, and as few as 310 zeptomoles (3.1 × 10-19 mol) were detectable in single droplets (8.8 nL). When applied to free fatty acid (FFA) uptake in 3T3-L1 adipocytes, this µChopper permitted single-cell FFA uptake rates to be quantified at 3.5 ± 0.2 × 10-15 mol cell-1 for the first time. Additionally, homogeneous immunoassays in droplets exhibited insulin detection limits of 9.3 nM or 190 amol (1.9 × 10-16 mol). The combination of this novel, automated µChopper with lock-in detection provides a high-performance platform for detecting small differences with standard fluorescence optics, particularly in situations where sample volume is limited. The technique should be simple to implement into a variety of other droplet fluidics devices.
Assuntos
Automação , Ácidos Graxos/análise , Fluorescência , Técnicas Analíticas Microfluídicas , Imagem Óptica , Células 3T3-L1 , Adipócitos/química , Adipócitos/metabolismo , Animais , Ácidos Graxos/metabolismo , Camundongos , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
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.