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

Microfluidic 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.

Multiple Heart-Cutting Two-Dimensional Chip-HPLC Combined with Deep-UV Fluorescence and Mass Spectrometric Detection.

In this work, we introduce a new two-dimensional chip-based high-performance liquid chromatography (2D chip-HPLC) approach, which enables multiple transfers from the first dimension effluent onto the column head of the second separation dimension. By merging injection, separation, and detection features on a fused silica chip in a dead volume-free manner, all extra-column peak dispersion effects can be reduced to an absolute minimum. The application of intrinsic fluorescence detection with excitation in the deep-UV spectral region and electrospray ionization mass spectrometry after the first and second separation dimension, respectively, enables the label-free analysis of complex samples, as exemplarily shown for a pesticide mixture and a tryptic digest.

A microfluidic device enabling surface-enhanced Raman spectroscopy at chip-integrated multifunctional nanoporous membranes.

A three-dimensional microfluidic chip that combines sample manipulation and SERS detection on-chip was developed. This was successfully achieved by chip integration of a nanoporous polycarbonate track-etched (PCTE) membrane which connects microfluidic channels on two different levels with each other. The membrane fulfills two functions at the same time. On the one hand, it enables sample enrichment by selective electrokinetic transport processes through the membrane. On the other hand, the silver nanoparticle-coated backside of the same membrane enables SERS detection of the enriched analytes. The SERS substrate performance and the electrokinetic transport phenomena were studied using Rhodamine B (RhB) by Raman microscopy and fluorescence video microscopy. After system validation, the approach was attested by on-chip processing of a complex food sample. In a proof-of-concept study, the microfluidic device with the SERS substrate membrane was used to detect a concentration of 1 ppm melamine (705 cm-1) in whole milk. Electrokinetic transport across the nanoporous SERS substrate facilitates the extraction of analyte molecules from a sample channel into a detection channel via a potential gradient, thus easily removing obscuring compounds present in the sample matrix. The SERS signal of the analyte could be significantly increased by on-target sample drying. This was achieved by guiding an additional gas flow over the membrane which further extends the microfluidic functionality of the chip device. The proposed method possesses the advantages of combining a rapid (within 15 min) sample clean-up using electrokinetic transport in a three-dimensional microfluidic device which is highly suitable for sensitive and selective SERS detection of chemical and biological analytes. Graphical Abstract.

Nonaqueous Micro Free-Flow Electrophoresis for Continuous Separation of Reaction Mixtures in Organic Media.

The continuous separation mechanism of micro free-flow electrophoresis (µFFE) is a straightforward, suitable tool for microscale purification of reaction mixtures. However, aqueous separation buffers and organic reaction solvents limit the applicability of this promising combination. Herein, we have explored nonaqueous micro free-flow electrophoresis for this purpose and present its suitability for a continuous workup of organic reactions performed in acetonitrile. After successful nonaqueous FFE separation of organic dyes, the approach was applied to continuously recover the photocatalyst [Ru(bpy)3]2+ from a homogeneous, acetonitrile-based reaction mixture. This approach opens up possibilities for further downstream processing of purified products and is also attractive for recycling of precious catalyst species.

Supercritical-Fluid Chromatography On-Chip with Two-Photon-Excited-Fluorescence Detection for High-Speed Chiral Separations.

Herein, we present the first example of microchip-based supercritical-fluid chromatography (SFC). A microfluidic-glass-chip platform with pressure and temperature control for fast and efficient on-column injection is described. This enabled fast and efficient separation of chiral and achiral compounds within seconds and also employed two-photon-excitated-fluorescence detection. Peak shapes were highly regular and symmetric even for linear flow rates over the packed microchip column in a range of up to 20 mm·s-1.

Fluorescence lifetime-activated droplet sorting in microfluidic chip systems.

We present a highly efficient microfluidic fluorescence lifetime-activated droplet sorting (FLADS) approach as a novel technology for droplet manipulation in lab-on-a-chip devices. In a proof-of-concept study, we successfully applied the approach to sort droplets containing two different fluorescent compounds on the basis of their corresponding fluorescence lifetime. Towards this end, a technical set-up was developed enabling on-the-fly fluorescence lifetime determination of passing droplets. The herein developed LabVIEW program enabled fast triggering of a downstream dielectrophoretic force sorting functionality depending on average fluorescence lifetimes of individual droplets. The approach worked reliably at individual substrate concentrations from 1 nM to 1 mM. This not only allowed reliable sorting of droplets containing species with different fluorescence lifetimes but also enabled differentiation of mixtures in individual droplets.

Thermomechanical processing of In-containing ß-type Ti-Nb alloys.

In this study, the effect of thermomechanical processing on microstructure evolution of the indium-containing ß-type Ti alloys (Ti-40Nb)-3.5In and (Ti-36Nb)-3.5In was examined. Both alloys show an increased ß-phase stability compared to binary alloys due to In additions. This leads to a reduced α''-phase fraction in the solution treated and recrystallized state in the case of (Ti-36Nb)-3.5In and to the suppression of stress-induced α'' formation and deformation twinning for (Ti-40Nb)-3.5In. The mechanical properties of the alloys were subsequently studied by quasistatic tensile tests in the recrystallized state, revealing reduced Young's modulus values of 58GPa ((Ti-40Nb)-3.5In) and 56GPa ((Ti-36Nb)-3.5In) compared to 60GPa as determined for Ti-40Nb. For both In-containing alloys the ultimate tensile strength is in the range of 560MPa. Due to the suppressed α'' formation, (Ti-40Nb)-3.5In exhibits a linear elastic deformation behavior during tensile loading together with a low Young's modulus and is therefore promising for load-bearing implants.

Two-photon excitation in chip electrophoresis enabling label-free fluorescence detection in non-UV transparent full-body polymer chips.

One of the most commonly employed detection methods in microfluidic research is fluorescence detection, due to its ease of integration and excellent sensitivity. Many analytes though do not show luminescence when excited in the visible light spectrum, require suitable dyes. Deep-ultraviolet (UV) excitation (<300 nm) allows label-free detection of a broader range of analytes but also mandates the use of expensive fused silica glass, which is transparent to UV light. Herein, we report the first application of label-free deep UV fluorescence detection in non-UV transparent full-body polymer microfluidic devices. This was achieved by means of two-photon excitation in the visible range (λex = 532 nm). Issues associated with the low optical transmittance of plastics in the UV range were successfully circumvented in this way. The technique was investigated by application to microchip electrophoresis of small aromatic compounds. Various polymers, such as poly(methyl methacrylate), cyclic olefin polymer, and copolymer as well as poly(dimethylsiloxane) were investigated and compared with respect to achievable LOD and ruggedness against photodamage. To demonstrate the applicability of the technique, the method was also applied to the determination of serotonin and tryptamine in fruit samples.

Rapid prototyping of electrochromatography chips for improved two-photon excited fluorescence detection.

In the present study, we introduce two-photon excitation at 532 nm for label-free fluorescence detection in chip electrochromatography. Two-photon excitation at 532 nm offers a promising alternative to one-photon excitation at 266 nm, as it enables the use of economic chip materials instead of fused silica. In order to demonstrate these benefits, one-photon and two-photon induced fluorescence detection are compared in different chip layouts and materials with respect to the achievable sensitivity in the detection of polycyclic aromatic hydrocarbons (PAHs). Customized chromatography chips with cover or bottom slides of different material and thickness are produced by means of a rapid prototyping method based on liquid-phase lithography. The design of thin bottom chips (180 µm) enables the use of high-performance immersion objectives with low working distances, which allows one to exploit the full potential of two-photon excitation for a sensitive detection. The developed method is applied for label-free analysis of PAHs separated on a polymer monolith inside polymer glass sandwich chips made from fused silica or soda-lime glass. The obtained limits of detection range from 40 nM to 1.95 µM, with similar sensitivities in fused silica thin bottom chips for one-photon and two-photon excitation. In deep-UV non- or less-transparent devices two-photon excitation is mandatory for label-free detection of aromatics with high sensitivity.

Label-free fluorescence detection of aromatic compounds in chip electrophoresis applying two-photon excitation and time-correlated single-photon counting.

In this study, we introduce time-resolved fluorescence detection with two-photon excitation at 532 nm for label-free analyte determination in microchip electrophoresis. In the developed method, information about analyte fluorescence lifetimes is collected by time-correlated single-photon counting, improving reliable peak assignment in electrophoretic separations. The determined limits of detection for serotonin, propranolol, and tryptophan were 51, 37, and 280 nM, respectively, using microfluidic chips made of fused silica. Applying two-photon excitation microchip separations and label-free detection could also be performed in borosilicate glass chips demonstrating the potential for label-free fluorescence detection in non-UV-transparent devices. Microchip electrophoresis with two-photon excited fluorescence detection was then applied for analyses of active compounds in plant extracts. Harmala alkaloids present in methanolic plant extracts from Peganum harmala could be separated within seconds and detected with on-the-fly determination of fluorescence lifetimes.

State variables monitoring by in situ multi-wavelength fluorescence spectroscopy in heterologous protein production by Pichia pastoris.

State variables throughout non-induced and induced cultivations of Pichia pastoris for the heterologous Rhizopus oryzae lipase (ROL) production were monitored with a multi-wavelength on-line fluorescence sensor. Based on this work, the use of in situ multi-wavelength fluorometry combined with chemometrics models (PLS-1 models) provided a quantitative prediction of biomass and substrates (glycerol and methanol) during non-induced and induced ROL production. The mean prediction errors for both variables were about 7% and 10%, respectively. ROL is also quite satisfactory estimated in the exponential growth phase with prediction errors similar to biomass and substrate variables. However, in the stationary phase, where proteolytic degradation of ROL is observed, the prediction error could get a value about 20%. This fact is due to the lower reproducibility of protein production from batch to batch.