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

Droplet-based microfluidics has been widely applied in enzyme directed evolution (DE), in either cell or cell-free system, due to its low cost and high throughput. As the isolation principles are based on the labeled or label-free characteristics in the droplets, sorting method contributes mostly to the efficiency of the whole system. Fluorescence-activated droplet sorting (FADS) is the mostly applied labeled method but faces challenges of target enzyme scope. Label-free sorting methods show potential to greatly broaden the microfluidic application range. Here, we review the developments of droplet sorting methods through a comprehensive literature survey, including labeled detections [FADS and absorbance-activated droplet sorting (AADS)] and label-free detections [electrochemical-based droplet sorting (ECDS), mass-activated droplet sorting (MADS), Raman-activated droplet sorting (RADS), and nuclear magnetic resonance-based droplet sorting (NMR-DS)]. We highlight recent cases in the last 5 years in which novel enzymes or highly efficient variants are generated by microfluidic DE. In addition, the advantages and challenges of different sorting methods are briefly discussed to provide an outlook for future applications in enzyme DE.

Download Full-text

Two-photon fluorescence lifetime for label-free microfluidic droplet sorting

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-021-03745-2 ◽

2021 ◽

Author(s):

Sadat Hasan ◽

Maximilian E. Blaha ◽

Sebastian K. Piendl ◽

Anish Das ◽

David Geissler ◽

...

Keyword(s):

Fluorescence Lifetime ◽

Single Photon ◽

Photon Counting ◽

Droplet Microfluidics ◽

Label Free ◽

Single Photon Counting ◽

Two Photon ◽

Deep Uv ◽

Droplet Sorting ◽

532 Nm

AbstractMicrofluidic droplet sorting systems facilitate automated selective micromanipulation of compartmentalized micro- and nano-entities in a fluidic stream. Current state-of-the-art droplet sorting systems mainly rely on fluorescence detection in the visible range with the drawback that pre-labeling steps are required. This limits the application range significantly, and there is a high demand for alternative, label-free methods. Therefore, we introduce time-resolved two-photon excitation (TPE) fluorescence detection with excitation at 532 nm as a detection technique in droplet microfluidics. This enables label-free in-droplet detection of small aromatic compounds that only absorb in a deep-UV spectral region. Applying time-correlated single-photon counting, compounds with similar emission spectra can be distinguished due to their fluorescence lifetimes. This information is then used to trigger downstream dielectrophoretic droplet sorting. In this proof-of-concept study, we developed a polydimethylsiloxane-fused silica (FS) hybrid chip that simultaneously provides a very high optical transparency in the deep-UV range and suitable surface properties for droplet microfluidics. The herein developed system incorporating a 532-nm picosecond laser, time-correlated single-photon counting (TCSPC), and a chip-integrated dielectrophoretic pulsed actuator was exemplarily applied to sort droplets containing serotonin or propranolol. Furthermore, yeast cells were screened using the presented platform to show its applicability to study cells based on their protein autofluorescence via TPE fluorescence lifetime at 532 nm. Graphical abstract

Download Full-text

Positive dielectrophoresis–based Raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo

Science Advances ◽

10.1126/sciadv.abb3521 ◽

2020 ◽

Vol 6 (32) ◽

pp. eabb3521 ◽

Cited By ~ 2

Author(s):

Xixian Wang ◽

Yi Xin ◽

Lihui Ren ◽

Zheng Sun ◽

Pengfei Zhu ◽

...

Keyword(s):

Fatty Acid ◽

Signal Quality ◽

Label Free ◽

Theoretical Limit ◽

Degree Of Unsaturation ◽

Conflicting Demands ◽

Low Levels ◽

Droplet Sorting ◽

Positive Dielectrophoresis

The potential of Raman-activated cell sorting (RACS) is inherently limited by conflicting demands for signal quality and sorting throughput. Here, we present positive dielectrophoresis–based Raman-activated droplet sorting (pDEP-RADS), where a periodical pDEP force was exerted to trap fast-moving cells, followed by simultaneous microdroplet encapsulation and sorting. Screening of yeasts for triacylglycerol (TAG) content demonstrated near-theoretical-limit accuracy, ~120 cells min−1 throughput and full-vitality preservation, while sorting fatty acid degree of unsaturation (FA-DU) featured ~82% accuracy at ~40 cells min−1. From a yeast library expressing algal diacylglycerol acyltransferases (DGATs), a pDEP-RADS run revealed all reported TAG-synthetic variants and distinguished FA-DUs of enzyme products. Furthermore, two previously unknown DGATs producing low levels of monounsaturated fatty acid–rich TAG were discovered. This first demonstration of RACS for enzyme discovery represents hundred-fold saving in time consumables and labor versus culture-based approaches. The ability to automatically flow-sort resonance Raman–independent phenotypes greatly expands RACS’ application.

Download Full-text

A General Method for Spectrometer Design in the Scoff Approximation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100092657 ◽

1976 ◽

Vol 34 ◽

pp. 538-539

Author(s):

J. R. Fields

Keyword(s):

Electron Microscope ◽

Transmission Electron Microscope ◽

Energy Analysis ◽

Incident Beam ◽

Scanning Transmission Electron Microscope ◽

Chemical Identification ◽

Scanning Transmission Electron ◽

Transmission Electron ◽

Scanning Transmission ◽

General Method

The energy analysis of electrons scattered by a specimen in a scanning transmission electron microscope can improve contrast as well as aid in chemical identification. In so far as energy analysis is useful, one would like to be able to design a spectrometer which is tailored to his particular needs. In our own case, we require a spectrometer which will accept a parallel incident beam and which will focus the electrons in both the median and perpendicular planes. In addition, since we intend to follow the spectrometer by a detector array rather than a single energy selecting slit, we need as great a dispersion as possible. Therefore, we would like to follow our spectrometer by a magnifying lens. Consequently, the line along which electrons of varying energy are dispersed must be normal to the direction of the central ray at the spectrometer exit.

Download Full-text

Label-free, mass-sensitive single-molecule imaging using interferometric scattering microscopy

Essays in Biochemistry ◽

10.1042/ebc20200023 ◽

2020 ◽

Author(s):

Nikolas Hundt

Keyword(s):

Single Molecule ◽

Cellular Systems ◽

Single Molecule Imaging ◽

Label Free ◽

Measurement Sensitivity ◽

Mass Distributions ◽

Quantitative Measurements ◽

Contrast Mechanism ◽

Cellular Components ◽

Scattering Signal

Abstract Single-molecule imaging has mostly been restricted to the use of fluorescence labelling as a contrast mechanism due to its superior ability to visualise molecules of interest on top of an overwhelming background of other molecules. Recently, interferometric scattering (iSCAT) microscopy has demonstrated the detection and imaging of single biomolecules based on light scattering without the need for fluorescent labels. Significant improvements in measurement sensitivity combined with a dependence of scattering signal on object size have led to the development of mass photometry, a technique that measures the mass of individual molecules and thereby determines mass distributions of biomolecule samples in solution. The experimental simplicity of mass photometry makes it a powerful tool to analyse biomolecular equilibria quantitatively with low sample consumption within minutes. When used for label-free imaging of reconstituted or cellular systems, the strict size-dependence of the iSCAT signal enables quantitative measurements of processes at size scales reaching from single-molecule observations during complex assembly up to mesoscopic dynamics of cellular components and extracellular protrusions. In this review, I would like to introduce the principles of this emerging imaging technology and discuss examples that show how mass-sensitive iSCAT can be used as a strong complement to other routine techniques in biochemistry.

Download Full-text

A microfluidic device with integrated impedance detection for λ-DNA

MRS Proceedings ◽

10.1557/proc-773-n6.5 ◽

2003 ◽

Vol 773 ◽

Cited By ~ 1

Author(s):

Myung-Il Park ◽

Jonging Hong ◽

Dae Sung Yoon ◽

Chong-Ook Park ◽

Geunbae Im

Keyword(s):

Microfluidic Device ◽

Electrochemical Impedance ◽

Direct Detection ◽

Full Potential ◽

Label Free ◽

Buffer Solution ◽

Detection Systems ◽

Significant Difference ◽

Detection Of Biomolecules ◽

Impedance Magnitude

AbstractThe large optical detection systems that are typically utilized at present may not be able to reach their full potential as portable analysis tools. Accurate, early, and fast diagnosis for many diseases requires the direct detection of biomolecules such as DNA, proteins, and cells. In this research, a glass microchip with integrated microelectrodes has been fabricated, and the performance of electrochemical impedance detection was investigated for the biomolecules. We have used label-free λ-DNA as a sample biomolecule. By changing the distance between microelectrodes, the significant difference between DW and the TE buffer solution is obtained from the impedance-frequency measurements. In addition, the comparison for the impedance magnitude of DW, the TE buffer, and λ-DNA at the same distance was analyzed.

Download Full-text