Configurable 3D Printed Microfluidic Multiport Valves with Axial Compression

In the last decade, the fabrication of microfluidic chips was revolutionized by 3D printing. It is not only used for rapid prototyping of molds, but also for manufacturing of complex chips and even integrated active parts like pumps and valves, which are essential for many microfluidic applications. The manufacturing of multiport injection valves is of special interest for analytical microfluidic systems, as they can reduce the injection to detection dead volume and thus enhance the resolution and decrease the detection limit. Designs reported so far use radial compression of rotor and stator. However, commercially available nonprinted valves usually feature axial compression, as this allows for adjustable compression and the possibility to integrate additional sealing elements. In this paper, we transfer the axial approach to 3D-printed valves and compare two different printing techniques, as well as six different sealing configurations. The tightness of the system is evaluated with optical examination, weighing, and flow measurements. The developed system shows similar performance to commercial or other 3D-printed valves with no measurable leakage for the static case and leakages below 0.5% in the dynamic case, can be turned automatically with a stepper motor, is easy to scale up, and is transferable to other printing methods and materials without design changes

Micro simulated moving bed chromatography-mass spectrometry as a continuous on-line process analytical tool

Continuous manufacturing is becoming increasingly important in the (bio-)pharmaceutical industry, as more product can be produced in less time and at lower costs. In this context, there is a need for powerful continuous analytical tools. Many established off-line analytical methods, such as mass spectrometry (MS), are hardly considered for process analytical technology (PAT) applications in biopharmaceutical processes, as they are limited to at-line analysis due to the required sample preparation and the associated complexity, although they would provide a suitable technique for the assessment of a wide range of quality attributes. In this study, we investigated the applicability of a recently developed micro simulated moving bed chromatography system (µSMB) for continuous on-line sample preparation for MS. As a test case, we demonstrate the continuous on-line MS measurement of a protein solution (myoglobin) containing Tris buffer, which interferes with ESI-MS measurements, by continuously exchanging this buffer with a volatile ammonium acetate buffer suitable for MS measurements. The integration of the µSMB significantly increases MS sensitivity by removing over 98% of the buffer substances. Thus, this study demonstrates the feasibility of on-line µSMB-MS, providing a versatile PAT tool by combining the detection power of MS for various product attributes with all the advantages of continuous on-line analytics

Automated Solid-Phase Protein Modification with Integrated Enzymatic Digest for Reaction Validation: Application of a Compartmented Microfluidic Reactor for Rapid Optimization and Analysis of Protein Biotinylation

Protein modification by covalent coupling of small ligands or markers is an important prerequisite for the use of proteins in many applications. Well-known examples are the use of proteins with fluorescent markers in many in vivo experiments or the binding of biotinylated antibodies via biotin–streptavidin coupling in the frame of numerous bioassays. Multiple protocols were established for the coupling of the respective molecules, e.g., via the C and N-terminus, or via cysteines and lysines exposed at the protein surface. Still, in most cases the conditions of these standard protocols are only an initial guess. Optimization of the coupling parameters like reagent concentrations, pH, or temperature may strongly increase coupling yield and the biological activity of the modified protein. In order to facilitate the process of optimizing coupling conditions, a method was developed which uses a compartmented microfluidic reactor for the rapid screening of different coupling conditions. In addition, the system allows for the integration of an enzymatic digest of the modified protein directly after modification. In combination with a subsequent MALDI-TOF analysis of the resulting fragments, this gives a fast and detailed picture not only of the number and extent of the generated modifications but also of their position within the protein sequence. The described process was demonstrated for biotinylation of green fluorescent protein. Different biotin-excesses and different pH-values were tested in order to elucidate the influence on the modification extent and pattern. In addition, the results of solid-phase based modifications within the microfluidic reactor were compared to modification patterns resulting from coupling trials with unbound protein. As expected, modification patterns of immobilized proteins showed clear differences to the ones of dissolved proteins

Automated Solid-Phase Protein Modification with Integrated Enzymatic Digest for Reaction Validation: Application of a Compartmented Microfluidic Reactor for Rapid Optimization and Analysis of Protein Biotinylation

Protein modification by covalent coupling of small ligands or markers is an important prerequisite for the use of proteins in many applications. Well-known examples are the use of proteins with fluorescent markers in many in vivo experiments or the binding of biotinylated antibodies via biotin–streptavidin coupling in the frame of numerous bioassays. Multiple protocols were established for the coupling of the respective molecules, e.g., via the C and N-terminus, or via cysteines and lysines exposed at the protein surface. Still, in most cases the conditions of these standard protocols are only an initial guess. Optimization of the coupling parameters like reagent concentrations, pH, or temperature may strongly increase coupling yield and the biological activity of the modified protein. In order to facilitate the process of optimizing coupling conditions, a method was developed which uses a compartmented microfluidic reactor for the rapid screening of different coupling conditions. In addition, the system allows for the integration of an enzymatic digest of the modified protein directly after modification. In combination with a subsequent MALDI-TOF analysis of the resulting fragments, this gives a fast and detailed picture not only of the number and extent of the generated modifications but also of their position within the protein sequence. The described process was demonstrated for biotinylation of green fluorescent protein. Different biotin-excesses and different pH-values were tested in order to elucidate the influence on the modification extent and pattern. In addition, the results of solid-phase based modifications within the microfluidic reactor were compared to modification patterns resulting from coupling trials with unbound protein. As expected, modification patterns of immobilized proteins showed clear differences to the ones of dissolved proteins

Safety, feasibility, and early efficacy of the water-specific 1940-nm laser wavelength for ablation of saphenous incompetence

Objective: The aim of the present study was to evaluate the safety, feasibility, and early efficacy of saphenous vein ablation using a water-specific 1940-nm diode laser wavelength using low linear endovenous energy density. Methods: We retrospectively analyzed a series of patients who had undergone endovenous laser ablation (EVLA) between July 2020 and October 2021 from the multicenter, prospectively maintained VEINOVA (vein occlusion with various techniques) registry. EVLA was performed using a water-specific 1940-nm radial laser fiber. In the same session, all insufficient tributaries were treated by phlebectomy or sclerotherapy. Tumescent anesthesia was injected into the perivenous space. The vein diameter, energy delivered, and linear endovenous density were reviewed at baseline. The incidence of venous thromboembolism, endovenous heat-induced thrombosis (EHIT), burns, phlebitis, paresthesia, and occlusion were reviewed at 2 days and 6 weeks of follow-up. We used descriptive statistics to describe the results. Results: Overall, 229 patients were identified. Of the 229 patients, 34 were excluded because of treatment of recurrent varicose veins at a previously operated site (residual or neovascularization). Finally, 108 patients with varicose veins and 87 with recurrent varicose veins (new varicose veins in an untreated area) due to disease progression were included in the present analysis. A total of 256 native saphenous veins (163 great saphenous veins, 53 small saphenous veins, and 40 accessory saphenous veins) in 224 legs had undergone EVLA. The mean patient age was 58.3 ± 16.5 years. Of the 195 patients, 134 (68.7%) were women and 61 (31.3%) were men. Nearly one half of the patients had a history of saphenous vein surgery (44.6%). The CEAP (clinical, etiology, anatomy, pathophysiology) class was C2 in 31 legs (13.8%), C3 in 108 (48.2%), C4a to C4c in 72 (32.1%), and C5 or C6 in 13 legs (5.8%). The treatment length was 34.8 ± 18.3 cm. The mean diameter was 5.0 ± 1.2 mm. The average linear endovenous density was 34.8 ± 9.2 J/cm. Concomitant miniphlebectomy was performed in 163 patients (83.6%) and concomitant sclerotherapy in 35 patients (18%). At 2 days and 6 weeks of follow-up, the occlusion rate of the treated truncal veins was 99.6% and 99.6%, respectively, with only one truncal vein (0.4%) with partial recanalization at 2 days and 6 weeks of follow-up. No cases of proximal deep vein thrombosis, pulmonary embolism, or EHIT had occurred at follow-up. Only one patient (0.5%) had developed calf deep vein thrombosis at 6 weeks of follow-up. The incidence of postoperative ecchymosis was rare (1.5%) and had resolved at 6 weeks of follow-up. Conclusions: EVLA of incompetent saphenous veins using the water-specific 1940-nm diode laser wavelength is feasible and appears to be safe and efficient with a high occlusion rate, minimal side effects, and a zero rate of EHIT