Broad-Band Spectrum, High-Sensitivity Absorbance Spectroscopy in Picoliter Volumes

Abstract

Picoliter-volume droplets within segmented flows can be probed in a rapid and efficient manner using optical detection methods. To date, however, most detection schemes for droplet content analysis have relied on the use of time-integrated fluorescence measurements. Despite its undoubted utility, the implementation of absorbance-based detectors is particularly challenging due to the reduced optical path lengths that are characteristic of microfluidic systems and deleterious scattering at droplet–oil interfaces. Unsurprisingly, efforts to develop sensitive absorbance-based detection schemes for the interrogation of rapidly moving droplets have primarily focused on ensuring adequate analytical sensitivity and, to date, have been exclusively limited to single-wavelength measurements. To address this limitation, and expand the information content associated with absorbance measurements on-chip, we herein describe a detection scheme for the extraction of broad-band absorbance spectra from pL-volume droplets with high sensitivity. The combination of a confocal optical system (that confines incident light to a reduced detection volume) and a postprocessing algorithm (that effectively removes the contribution of the carrier oil from the extracted spectra) engenders significant improvements in signal-to-noise ratios. Our system is initially calibrated by acquiring absorbance spectra from aqueous solutions of fluorescein isothiocyanate. These measurements confirm both excellent linearity over the studied range (from 0 to 100 μM) and a concentration limit of detection of 800 nM. The methodology is then used to monitor the salt-induced aggregation of gold nanoparticles with millisecond time resolution. This approach for small-volume absorbance spectroscopy allows for both high-throughput and high-information content measurements in subnanoliter volumes and will be highly desirable in a wide variety of bioanalytical applications where sensitivity and throughput are priorities.

Plasmonic Amyloid Tactoids

“…The laser lines were chosen to best excite the intrinsic luminescence of the AFs as well as to match with the SPR frequencies of the GNRs. The polarization‐dependent extinction spectra of the hybrid tactoids were measured by confining the polarized illumination with a pinhole using a homebuilt setup, [ 40 ] enabling the confocal illumination of single tactoids. The reference of the extinction measurement was collected from the isotropic surroundings, which contains less concentrated GNRs as indicated by the spectra.…”

Sorting of droplets at kHz rates using absorbance activated acoustic sorting

“…3,4,8,9,12 The presence of a fluorophore label within the droplets is required to produce the fluorescent readout, thus introducing an artefact into the system. 2 Fluorescent sorting devices usually require expensive and complex optical set-ups 13 . In contrast, absorbance detection has the potential for smaller and simpler set-ups as well as not requiring a fluorescent label.…”

“…20 A challenge with absorbance detection in droplets is their small volume (pL), and the resulting short optical path length (μm), which has an impact on the sensitivity of the detection. 2,14 Absorbance can be detected using integrated optic fibres. The voltage signals corelate to Beer-Lamberts Law and the droplet solutions absorbance can be calculated.…”

“…For their directed evolution experiment 1 mM WST1-formazan was added to the assay to mask the artefacts in the signal. 12 More recently, full UV-Vis spectra screening and sorting has been demonstrated by Probst et al 2 and Duncombe et al 15 with achieved droplet rates below 100 Hz since full spectra screening decreases throughput. Duncombe et al 15 incorporated an increased path length from 50 to 300 μm and droplet volumes above 300 pL.…”

UV–Vis Spectra-Activated Droplet Sorting for Label-Free Chemical Identification and Collection of Droplets

“…Analytically, this is particularly useful at UVB and UVC wavelengths (<315 nm), where DNA, aromatic amino acids, and other unique chemical signatures can be observed . There have been several published microfluidic devices that utilize droplet absorbance measurements ,, but none, to date, that incorporate high-throughput full-spectrum UV–vis spectroscopy in screening assays. Gielen et al developed an absorbance-activated droplet sorter for the use of colorimetric enzymatic assays with integrated optical fibers, a single-wavelength visible-range LED light source, and a photodiode.…”