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Design and operation of the microPAD. (A) Design of the device. (B) Operation of the device flows through (a) loading and metering, (b) pushing in, and (c) mixing. (C) (a) Design of a mixing valve and (b) operation of the mixing valves.

Design and operation of the microPAD. (A) Design of the device. (B) Operation of the device flows through (a) loading and metering, (b) pushing in, and (c) mixing. (C) (a) Design of a mixing valve and (b) operation of the mixing valves.

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Article
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A microfluidic protein aggregation device (microPAD) that allows the user to perform a series of protein incubations with various concentrations of two reagents is demonstrated. The microfluidic device consists of 64 incubation chambers to perform individual incubations of the protein at 64 specific conditions. Parallel processes of metering reagen...

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... device consists of 64 incubation units. Each unit consists of a pushing line, a metering unit, and an incubation chamber ( Figure 1A,B). Figure 1B shows the step-by-step operation of the device. ...
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... unit consists of a pushing line, a metering unit, and an incubation chamber ( Figure 1A,B). Figure 1B shows the step-by-step operation of the device. The operation steps include (1) loading the reagents, (2) pushing the metered reagents into reaction chambers, and (3) mixing the reagents by using mixing valves located in the center of the chamber (Movie S1 in the Supplementary Materials). ...
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... metering unit comprises four loading sites: a dilution solution site (yellow color), a factor #1 site (blue color), a factor #2 site (red color), and a main factor site (green color). The samples were loaded by pressurizing them from the inlets while the central valves were closed and the side valves in the metering units were open ( Figure 1B(a)). The metering units were designed to create stepwise gradients of two reagents, i.e., in the ratios of 1:1, 1:1.57, 1:2.13, 1:2.7, 1:3.27, 1:3.83, 1:4.4, ...
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... 1:4.97, for each reagent (Table 1). After the metering of the reagents, the central valves were closed, the side valves were opened, and the reagents were pushed into the incubation chambers ( Figure 1B(b)). Then, all valves were closed, and the reagents were mixed by the mixing valves ( Figure 1B(c)). ...
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... the metering of the reagents, the central valves were closed, the side valves were opened, and the reagents were pushed into the incubation chambers ( Figure 1B(b)). Then, all valves were closed, and the reagents were mixed by the mixing valves ( Figure 1B(c)). Figure 1C shows the design and operation of the mixing valves. ...
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... all valves were closed, and the reagents were mixed by the mixing valves ( Figure 1B(c)). Figure 1C shows the design and operation of the mixing valves. The valves were designed to push up a certain volume at the center of the incubation chamber by actuation of the membrane between a fluidic channel and a control channel ( Figure 1C(a)) [39,43,44]. ...
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... 1C shows the design and operation of the mixing valves. The valves were designed to push up a certain volume at the center of the incubation chamber by actuation of the membrane between a fluidic channel and a control channel ( Figure 1C(a)) [39,43,44]. The actuation height of the membrane is controlled by a pressure applied via the control channel, as was shown by simulation and testing ( Figure S1 in the Supplementary Materials). ...
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... valves were designed to push up a certain volume at the center of the incubation chamber by actuation of the membrane between a fluidic channel and a control channel ( Figure 1C(a)) [39,43,44]. The actuation height of the membrane is controlled by a pressure applied via the control channel, as was shown by simulation and testing ( Figure S1 in the Supplementary Materials). The optimal pressure and operating frequency of the valve to mix reagents in the chamber were determined to be 0.2 bar and 1.0 Hz, respectively. ...
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... optimal pressure and operating frequency of the valve to mix reagents in the chamber were determined to be 0.2 bar and 1.0 Hz, respectively. Microscope images in Figure 1C(b) show de-actuation (top) and actuation (bottom) of four mixing valves. The operation of 8 mixing valves is shown in Movie S2 in the Supplementary Materials. ...

Citations

... The chains are connected by two disulphide bridges located between Cys-A7 and Cys-B7 as well as Cys-A20 and Cys-B19. In the A chain, there is a third disulphide bond linking Cys-A6 and Cys-A11 [2,3]. Insulin plays an important role in maintaining whole-body homeostasis. ...
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Insulin loaded to the polymer network of hydrogels may affect the speed and the quality of wound healing in diabetic patients. The aim of our research was to develop a formulation of insulin that could be applied to the skin. We chose hydrogels commonly used for pharmaceutical compounding, which can provide a form of therapy available to every patient. We prepared different gel formulations using Carbopol® UltrezTM 10, Carbopol® UltrezTM 30, methyl cellulose, and glycerin ointment. The hormone concentration was 1 mg/g of the hydrogel. We assessed the influence of model hydrogels on the pharmaceutical availability of insulin in vitro, and we examined the rheological and the texture parameters of the prepared formulations. Based on spectroscopic methods, we evaluated the influence of model hydrogels on secondary and tertiary structures of insulin. The analysis of rheograms showed that hydrogels are typical of shear-thinning non-Newtonian thixotropic fluids. Insulin release from the formulations occurs in a prolonged manner, providing a longer duration of action of the hormone. The stability of insulin in hydrogels was confirmed. The presence of model hydrogel carriers affects the secondary and the tertiary structures of insulin. The obtained results indicate that hydrogels are promising carriers in the treatment of diabetic foot ulcers. The most effective treatment can be achieved with a methyl cellulose-based insulin preparation.
... The microfluidic chip was fabricated by multilayer soft lithography using polydimethylsiloxane (PDMS) [10,29], following the modified protocols from our previous work [14,[30][31][32]. ...
... The droplets were collected in the incubation stages, and the RD fluorescent signal of the droplets was quantified by N 2.1 filter cube (excitation: BP 515-560 nm; emission: LP 590 nm). An indium tin oxide (ITO) heater and a controller were obtained from Cell MicroControls (Norfolk, VA, USA) and calibrated to vary the temperature in the incubation stages in the microfluidic device [32]. Note that the temperature was changed after performing a serial dilution in a series of droplets, so potential changes in solution viscosity or PDMS elasticity due to the temperature change, which could affect droplet size and dilution, is avoided. ...
... This maximum decrease rate is in close agreement with values reported in the literature of 1.90 %/ C, which was measured for rhodamine B in water using a spectrofluorometer equipped with a temperature control module [37]. The reliable fluorometric measurement of molecules in droplets associated with the fast heat transfer in such small volumes, providing exquisite control on the temperature is of great interest to monitor the temperature-dependent kinetics of reactions with fluorescence markers for biotechnological studies, e.g., protein aggregation [32,38], and enzyme kinetics [14,23]. ...
Article
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A programmable droplet-based microfluidic serial dilutor platform is presented, which is capable of generating a series of droplets with the scalable stepwise concentration gradient of a sample. Sequential dilution of a target molecule was automatically performed in sub-nanoliter scale droplets by synchronizing a microfluidic peristaltic mixer and a valve-assisted droplet generator. The volume of droplets dispensed from the mixer was controlled by microvalve operation, which enabled to tune the dilution with various dilution factors. After evaluation of the mixer efficiency and calibration of the droplet size at different valve operating conditions, serial dilutions of rhodamine B isothiocyanate-dextran was demonstrated, in an automated manner, at three different dilution factors. Specifically, the effect of the rhodamine B isothiocyanate-dextran concentration and temperature on variations of the fluorescent intensity was quantified. This programmable microfluidic droplet serial dilutor will open new avenues, an analytical tool, to evaluate complex chemical and biochemical reactions, especially when limited sample volume is available, for example, at the early stage of drug discovery and biochemical process developing.
... The platforms showed the potential of the valve-assisted droplet generator to produce highly monodispersed droplets and combinatorial contents in a series of droplets. Furthermore, the accurate manipulation of complex fluid flows by multilayer devices, where tens-or hundreds of microvalves were integrated, in previous reports [35][36][37] showed promise to engineer an automated, multifunctional microfluidic droplet array. ...
... The microfluidic device was fabricated by multilayer soft lithography technique [31,39], and we followed a modified fabrication protocol based on our previous studies [36,37]. The PDMS device consisted of a top fluidic layer and a bottom control layer; the heights of fluid flow channels and control channels were 38 ± 2 μm and 18 ± 2 μm (n = 10), respectively. ...
Article
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A microfluidic droplet-storage array that is capable of the continuous operation of droplet formation, storing, repositioning, retrieving, injecting and restoring is demonstrated. The microfluidic chip comprised four valve-assisted droplet generators and a 3 × 16 droplet-storage array. The integrated pneumatically actuated microvalves enable the precise control of aqueous phase dispensing, as well as carrier fluid flow path and direction for flexible manipulating water-in-oil droplets in the chip. The size of droplets formed by the valve-assisted droplet generators was validated under various operating conditions such as pressures for introducing solutions and dispensing time. In addition, flexible droplet addressing in the storage array was demonstrated by storing droplets with various numbers and compositions in different storage units as well as rearranging their stored positions. Moreover, serial injections of new droplets into a retrieved droplet from a storage unit was performed to show the potential of the platform in sequential dosing on incubated droplet-based reactors at the desired timeline. The droplet-storage array with great freedom and flexibility in droplet handling could be applied for performing complex chemical and biologic reactions, especially in which incubation and dosing steps are necessary.
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Imaging is increasingly more utilized as analytical technology in biopharmaceutical formulation research, with applications ranging from subvisible particle characterization to thermal stability screening and residual moisture analysis. This review offers a comprehensive overview of analytical imaging for scientists active in biopharmaceutical formulation research and development, where it presents the unique information provided by the ultraviolet (UV), visible (Vis), and infrared (IR) sections in the electromagnetic spectrum. The main body of this review consists of an outline of UV, Vis, and IR imaging techniques for several (bio)physical properties that are commonly determined during protein-based biopharmaceutical formulation characterization and development studies. The review concludes with a future perspective of applied imaging within the field of biopharmaceutical formulation research.