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The mechanical heart valve is a crucial solution for many patients. However, it cannot function on the state of blood as human tissue valves. Thus, people with mechanical valves are put under anticoagulant therapy. A good measurement of the state of blood and how long it takes blood to form clots is the prothrombin time (PT); moreover, it is an indicator of how well the anticoagulant therapy is, and of whether the response of the patient to the drug is as needed. For a more specific standardized measurement of coagulation time, an international normalized ratio (INR) is established. Clinical testing of INR and PT is relatively easy. However, it requires the patient to visit the clinic for evaluation purposes. Many techniques are therefore being developed to provide PT and INR self-testing devices. Unfortunately, those solutions are either inaccurate, complex, or expensive. The present work approaches the design of an anticoagulation self-monitoring device that is easy to use, accurate, and relatively inexpensive. Hence, a two-channel polymethyl methacrylate-based microfluidic point-of-care (POC) smart device has been developed. The Arduino based lab-on-a-chip device applies optical properties to a small amount of blood. The achieved accuracy is 96.7%.
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Coeficiente Internacional Normatizado/instrumentação , Dispositivos Lab-On-A-Chip , Testes Imediatos , Tempo de Protrombina/instrumentação , Anticoagulantes/uso terapêutico , Biologia Computacional , Desenho de Equipamento , Próteses Valvulares Cardíacas , Humanos , Coeficiente Internacional Normatizado/métodos , Coeficiente Internacional Normatizado/estatística & dados numéricos , Dispositivos Lab-On-A-Chip/estatística & dados numéricos , Dispositivos Ópticos/estatística & dados numéricos , Testes Imediatos/estatística & dados numéricos , Polimetil Metacrilato , Tempo de Protrombina/métodos , Tempo de Protrombina/estatística & dados numéricos , AutotesteRESUMO
Glucose oxidase (GOx) is an enzyme with important industrial and biochemical applications, particularly in glucose detection. Here we leveraged the oxidative self-polymerization phenomenon of simple polyphenols (pyrogallol or catechol) in the presence of polyethylenimine (PEI) to form adhesive coatings that enabled GOx immobilization on conventional multi-well plates. Immobilization was verified and optimized by directly measuring GOx activity inside the coated wells. Our results showed that incorporating PEI in polyphenol coatings enhanced their enzyme immobilization efficiency, with pyrogallol (PG)-based coatings displaying the greatest enzyme activity. The immobilized enzyme maintained similar affinity to glucose compared to the free enzyme. GOx-immobilized PG/PEI-coated wells exhibited intermediate recycling ability but excellent resistance to urea as a denaturing agent compared to the free enzyme. GOx-immobilized 96-well plates allowed the construction of a linear glucose calibration curve upon adding glucose standards, with a detection limit of 0.4-112.6 mg dL-1, which was comparable to commercially available enzymatic glucose assay kits. The assay platform was also capable of effectively detecting glucose in rat plasma samples. Our findings present a simple enzyme immobilization technique that can be used to construct a glucose assay platform in a convenient multi-well format for high-throughput glucose quantification.
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In spite of the growing interest in the roles and applications of small RNAs (sRNAs), sRNA isolation methods are inconsistent, tedious, and dependent on the starting number of cells. In this work, we employ ITP to isolate sRNAs from the cell-lysate of K562 (chronic myelogenous leukemia) cells in a polydimethylsiloxane (PDMS) mesofluidic device. Our method specifically purifies sRNA of <60 nucleotides from lysate of a wide range of cell number spanning from 100 to 1 000 000 cells. We measured the amount of sRNA using the Agilent Bioanalyzer and further verified the extraction efficiency by reverse transcription quantitative PCR. Our method was shown to be more efficient in sRNA extraction than commercial sRNA isolation kits, especially when using smaller numbers of starting cells. Our assay presents a simple and rapid sRNA extraction method with 20 min assay time and no intermediate transfer steps.
Assuntos
Isotacoforese/métodos , Técnicas Analíticas Microfluídicas/métodos , Pequeno RNA não Traduzido/análise , Pequeno RNA não Traduzido/isolamento & purificação , Humanos , Células K562 , Pequeno RNA não Traduzido/químicaRESUMO
Blood viscosity measurements are crucial for the diagnosis and understanding of a range of hematological and cardiovascular diseases. Such measurements are heavily used in monitoring patients during and after surgeries, which necessitates the development of a highly accurate viscometer that uses a minimal amount of blood. In this work, we have designed and implemented a microfluidic device that was used to measure fluid viscosity with a high accuracy using less than 10 µl of blood. The device was further used to construct a blood viscosity model based on temperature, shear rate, and anti-coagulant concentration. The model has an R-squared value of 0.950. Finally, blood protein content was changed to simulate diseased conditions and blood viscosity was measured using the device and estimated using the model constructed in this work. Simulated diseased conditions were clearly detected when comparing estimated viscosity values using the model and the measured values using the device, proving the applicability of the setup in the detection of rheological anomalies and in disease diagnosis.
Assuntos
Viscosidade Sanguínea/efeitos dos fármacos , Heparina/farmacologia , Dispositivos Lab-On-A-Chip , Modelos Biológicos , Resistência ao Cisalhamento , Temperatura , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Dimetilpolisiloxanos , Relação Dose-Resposta a Droga , Desenho de Equipamento , NylonsRESUMO
We present an on-chip method for the extraction of RNA within a specific size range from low-abundance samples. We use isotachophoresis (ITP) with an ionic spacer and a sieving matrix to enable size-selection with a high yield of RNA in the target size range. The spacer zone separates two concentrated ITP peaks, the first containing unwanted single nucleotides and the second focusing RNA of the target size range (2-35 nt). Our ITP method excludes >90% of single nucleotides and >65% of longer RNAs (>35 nt). Compared to size selection using gel electrophoresis, ITP-based size-selection yields a 2.2-fold increase in the amount of extracted RNAs within the target size range. We also demonstrate compatibility of the ITP-based size-selection with downstream next generation sequencing. On-chip ITP-prepared samples reveal higher reproducibility of transcript-specific measurements compared to samples size-selected by gel electrophoresis. Our method offers an attractive alternative to conventional sample preparation for sequencing with shorter assay time, higher extraction efficiency and reproducibility. Potential applications of ITP-based size-selection include sequencing-based analyses of small RNAs from low-abundance samples such as rare cell types, samples from fluorescence activated cell sorting (FACS), or limited clinical samples.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Isotacoforese , RNA/química , RNA/isolamento & purificação , Linhagem Celular , Humanos , Íons/química , Dispositivos Lab-On-A-Chip , Tamanho da PartículaRESUMO
Although single-cell mRNA sequencing has been a powerful tool to explore cellular heterogeneity, the sequencing of small RNA at the single-cell level (sc-sRNA-seq) remains a challenge, as these have no consensus sequence, are relatively low abundant, and are difficult to amplify in a bias-free fashion. We present two methods of single-cell-lysis that enable sc-sRNA-seq. The first method is a chemical-based technique with overnight freezing while the second method leverages on-chip electrical lysis of plasma membrane and physical extraction and separation of cytoplasmic RNA via isotachophoresis. We coupled these two methods with off-chip small RNA library preparation using CleanTag modified adapters to prevent the formation of adapter dimers. We then demonstrated sc-sRNA-seq with single K562 human leukemic cells. Our approaches offer a relatively short hands-on time of 6 h and efficient generation of on-target reads. The sc-sRNA-seq with our approaches showed detection of miRNA with various abundances ranging from 16â¯000 copies/cell to about 10 copies/cell. We anticipate this approach will create a new opportunity to explore cellular heterogeneity through small RNA expression.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Pequeno RNA não Traduzido/genética , Análise de Célula Única/métodos , Estruturas Genéticas , Humanos , Células K562 , Dispositivos Lab-On-A-Chip , Octoxinol/química , Pequeno RNA não Traduzido/isolamento & purificação , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Análise de Célula Única/instrumentaçãoRESUMO
BACKGROUND: Improvement of medical content in Biomedical Engineering curricula based on a qualitative assessment process or on a comparison with another high-standard program has been approached by a number of studies. However, the quantitative assessment tools have not been emphasized. The quantitative assessment tools can be more accurate and robust in cases of challenging multidisciplinary fields like that of Biomedical Engineering which includes biomedicine elements mixed with technology aspects. The major limitations of the previous research are the high dependence on surveys or pure qualitative approaches as well as the absence of strong focus on medical outcomes without implicit confusion with the technical ones. The proposed work presents the development and evaluation of an accurate/robust quantitative approach to the improvement of the medical content in the challenging multidisciplinary BME curriculum. METHODS: The work presents quantitative assessment tools and subsequent improvement of curriculum medical content applied, as example for explanation, to the ABET (Accreditation Board for Engineering and Technology, USA) accredited biomedical engineering BME department at Jordan University of Science and Technology. The quantitative results of assessment of curriculum/course, capstone, exit exam, course assessment by student (CAS) as well as of surveys filled by alumni, seniors, employers and training supervisors were, first, mapped to the expected students' outcomes related to the medical field (SOsM). The collected data were then analyzed and discussed to find curriculum weakness points by tracking shortcomings in every outcome degree of achievement. Finally, actions were taken to fill in the gaps of the curriculum. Actions were also mapped to the students' medical outcomes (SOsM). RESULTS: Weighted averages of obtained quantitative values, mapped to SOsM, indicated accurately the achievement levels of all outcomes as well as the necessary improvements to be performed in curriculum. Mapping the improvements to SOsM also helps in the assessment of the following cycle. CONCLUSION: The suggested assessment tools can be generalized and extended to any other BME department. Robust improvement of medical content in BME curriculum can subsequently be achieved.
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Acreditação/normas , Educação de Pós-Graduação em Medicina , Avaliação Educacional/normas , Estudantes de Medicina , Engenharia Biomédica/normas , Currículo , Educação de Pós-Graduação em Medicina/normas , Humanos , Competência Profissional , Melhoria de QualidadeRESUMO
Parkinson's disease currently affects millions of people worldwide and is steadily increasing. Many symptoms are associated with this disease, including rest tremor, bradykinesia, stiffness or rigidity of the extremities and postural instability. No cure is currently available for Parkinson's disease patients; instead most medications are for treatment of symptoms. This treatment depends on the quantification of these symptoms such as hand tremor. This work proposes a new system for mobile phone applications. The system is based on measuring the acceleration from the Parkinson's disease patient's hand using a mobile cell phone accelerometer. Recordings from 21 Parkinson's disease patients and 21 healthy subjects were used. These recordings were analysed using a two level wavelet packet analysis and features were extracted forming a feature vector of 12 elements. The features extracted from the 42 subjects were classified using a neural networks classifier. The results obtained showed an accuracy of 95% and a Kappa coefficient of 90%. These results indicate that a cell phone accelerometer can accurately detect and record rest tremor in Parkinson's disease patients.
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Mãos/fisiopatologia , Aplicativos Móveis , Doença de Parkinson/fisiopatologia , Tremor/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Redes Neurais de Computação , Processamento de Sinais Assistido por ComputadorRESUMO
PDMS and PMMA are two of the most used polymers in the fabrication of lab-on-chip or microfluidic devices. In order to use these polymers in biological applications, it is sometimes essential to be able to bind biomolecules such as proteins and DNA to the surface of these materials. In this work, we have evaluated a number of processes that have been developed to bind protein to PDMS surfaces which include passive adsorption, passive adsorption with glutaraldehyde cross-linking, (3-aminopropyl) triethoxysilane functionalization followed by glutaraldehyde or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride cross-linkers. It has been shown that the latter technique--using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride--results in more than twice the bonding of protein to the surface of PDMS microchannels than proteins binding passively. We have also evaluated a few techniques that have been tested for the functionalization of PMMA microchannels where we have found that the use of polyethyleneimine (PEI) has led to the strongest protein-PMMA microchannel bond. We finally demonstrated the effect of PDMS curing methodology on protein adsorption to its surface, and showed that increased curing time is the factor that reduces passive adsorption the most.
Assuntos
Dimetilpolisiloxanos/química , Proteínas Imobilizadas/química , Técnicas Analíticas Microfluídicas/instrumentação , Polimetil Metacrilato/química , Técnicas Analíticas Microfluídicas/métodos , Propriedades de SuperfícieRESUMO
We have developed a microfluidic device that enhances the sensitivity of protein immunoassays by preconcentrating the protein sample using isotachophoresis (ITP). Two approaches were followed to study the sensitivity gain achieved that way. The first approach was using antibody-coated magnetic beads loaded into a microchannel to capture the proteins within the ITP sample zone. The second was to directly bind the antibodies to the microchannel surface. The use of ITP increased the sensitivity of both the direct and the bead-based immunoassay and lowered the model protein's detection limit. Our setup also uses electrokinetic injection for sample charging, thereby sparing the need for integrating mechanical components such as pumps or valves and reducing the complexity of the setup. This work demonstrates the feasibility of using ITP as a preconcentration mechanism of proteins in immunoassays. The protein was concentrated by a factor of 100 and the assay's limit of detection was in the picomolar range. ITP can be applied to any sample of mixed content, separating and preconcentrating the desired analyte before it reaches the detection region with immobilized antibodies, without the need for separating and concentrating the sample in an independent step.
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Anticorpos Imobilizados/química , Imunoensaio/instrumentação , Isotacoforese/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Soroalbumina Bovina/isolamento & purificação , Animais , Bovinos , Desenho de Equipamento , Limite de Detecção , Soroalbumina Bovina/análiseRESUMO
Cell-free protein synthesis (CFPS) enables rapid protein expression for the structural and functional characterization of proteins. Implementation of CFPS in a microfluidic platform has additional benefits such as reduced reaction volumes and simultaneous expression of multiple proteins. Here, we describe a microfluidic device that is composed of 96 continuous-exchange cell-free protein expression units and produces a protein synthesis yield up to 87 times higher than a conventional batch system.
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Técnicas Analíticas Microfluídicas/métodos , Biossíntese de Proteínas , Técnicas Analíticas Microfluídicas/instrumentação , Moldes Genéticos , Fatores de TempoRESUMO
Cell-free protein synthesis (CFPS) is an attractive alternative to cell-based protein expression systems because of its advantages including speed, simplicity, and adaptability to various formats. However, two major obstacles exist that have been preventing it from being widely used. One is high cost and the other is low protein synthesis yield. We report here a miniaturized CFPS device that addresses these challenges. The cost saving was achieved by miniaturization, which reduced the reagent consumption by two orders of magnitude. The protein synthesis yield was enhanced by prolonging CFPS reactions through continuous supply of reactants (e.g. nutrients and energy components). The reactants were contained in a feeding solution, which was replenished through a nanoporous membrane and microchannel. The design of the miniaturized device was optimized by running continuous-exchange CFPS in devices with a variation in the type of membrane, the size of the exchange interface, and the volume ratio of the reaction solution to the feeding solution. The effects of these design variations on the protein synthesis yield have been studied. Furthermore, the design was expanded into a 96-unit device that can produce a large number of proteins simultaneously, enabling high-throughput proteomics applications.
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Reatores Biológicos , Sistema Livre de Células , Membranas Artificiais , Miniaturização/instrumentação , Proteínas Recombinantes/metabolismo , Desenho de Equipamento , Luciferases , Miniaturização/métodos , Proteínas Recombinantes/análiseRESUMO
In this paper, we present a microfluidic platform for the continuous generation of stable, monodisperse lipid vesicles 20-110 µm in diameter. Our approach utilizes a microfluidic flow-focusing droplet generation design to control the vesicle size by altering the system's fluid flow rates to generate vesicles with narrow size distribution. Double emulsions are first produced in consecutive flow-focusing channel geometries and lipid membranes are then formed through a controlled solvent extraction process. Since no strong solvents are used in the process, our method allows for the safe encapsulation and manipulation of an assortment of biological entities, including cells, proteins, and nucleic acids. The vesicles generated by this method are stable and have a shelf life of at least 3 months. Here, we demonstrate the cell-free in vitro synthesis of proteins within lipid vesicles as an initial step towards the development of an artificial cell.
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Cell-free protein synthesis (CFPS) is an alternative approach to cell-based recombinant protein production. It involves in vitro transcription and translation in a cell-free medium. In this work, we implemented CFPS in a plastic array device. Each unit in the array consisted of an inner well and an outer well. Two synthesis steps, gene transcription and protein translation, took place in the inner well, in which a cell-free medium was used to provide ribosomes and additional components necessary for protein synthesis. The outer well was concentric to the inner well and it functioned as a nutrient reservoir. A nanoporous membrane was sandwiched between the inner and outer wells for retaining the synthesized proteins and removing the reaction byproducts. A microfluidic channel was employed to connect these two wells for supplying fresh nutrients for longer reaction time and higher expression yield. Synthesis of luciferase was shown to last 8 times longer and yield 10 times more proteins than in a conventional container. The device also enables more than 2 orders of magnitude reduction in reagent consumption compared to a bench-top instrument. The effects of the membrane pore size and microfluidic channel on the protein production yield were also studied. The array device has potential to become a platform for parallel protein expression for proteomics applications, matching high-throughput gene discovery.
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Sistema Livre de Células/química , Membranas Artificiais , Técnicas Analíticas Microfluídicas/instrumentação , Nanoestruturas/química , Nanotecnologia/instrumentação , Análise Serial de Proteínas/instrumentação , Proteínas/síntese química , Desenho de Equipamento , Análise de Falha de Equipamento , Nanoestruturas/ultraestrutura , PorosidadeRESUMO
Enzymes and membrane protein receptors represent almost three-quarters of all current drug targets. As a result, it would be beneficial to have a platform to produce them in a high-throughput format for drug screening. We have developed a miniaturized fluid array device for cell-free protein synthesis, and the device was exploited to produce both soluble and membrane proteins. Two membrane-associated proteins, bacteriorhodopsin and ApoA lipoprotein, were coexpressed in an expression medium in the presence of lipids. Simultaneous expression of ApoA lipoprotein enhanced the solubility of bacteriorhodopsin and would facilitate functional studies. In addition, the device was employed to produce two enzymes, luciferase and beta-lactamase, both of which were demonstrated to be compatible with enzyme inhibition assays. Beta-lactamase, a drug target associated with antibiotic resistance, was further used to show the capability of the device for drug screening. Beta-lactamase was synthesized in the 96 units of the device and then assayed by a range of concentrations of four mock drug compounds without harvesting and purification. The inhibitory effects of these compounds on beta-lactamase were measured in a parallel format, and the degree in their drug effectiveness agreed well with the data in the literature. This work demonstrated the feasibility of the use of the fluid array device and cell-free protein expression for drug screening, with advantages in less reagent consumption, shorter analysis time, and higher throughput.
Assuntos
Apolipoproteínas A/metabolismo , Bacteriorodopsinas/metabolismo , Análise Serial de Proteínas/métodos , Apolipoproteínas A/genética , Bacteriorodopsinas/genética , Sistema Livre de Células , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Cinética , Luciferases/antagonistas & inibidores , Luciferases/genética , Luciferases/metabolismo , Inibidores de beta-Lactamases , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
We describe a miniaturized fluid array device for high-throughput cell-free protein synthesis (CFPS), aiming to match the throughput and scale of gene discovery. Current practice of using E. coli cells for production of recombinant proteins is difficult and cost-prohibitive to implement in a high-throughput format. As more and more new genes are being identified, there is a considerable need to have high-throughput methods to produce a large number of proteins for studying structures and functions of the corresponding genes. The device consists of 96 units and each unit is for expression of one protein; thus up to 96 proteins can be produced simultaneously. The function of the fluid array was demonstrated by expression of a variety of proteins, with more than two orders of magnitude reduction in reagent consumption compared with a commercially available CFPS instrument. The protein expression yield in the device was up to 87 times higher for ß-glucoronidase than that in a conventional microplate. The concentration of ß-galactosidase expressed in the device was determined at 5.5 µg/µL. The feasibility of using the device for drug screening was demonstrated by measuring the inhibitory effects of mock drug compounds on synthesized ß-lactamase without the need for harvesting proteins, which enabled us to reduce the analysis time from days to hours.
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Fosfatase Alcalina/biossíntese , Ensaios de Triagem em Larga Escala/métodos , Luciferases/biossíntese , Análise Serial de Proteínas/métodos , beta-Galactosidase/biossíntese , beta-Lactamases/biossíntese , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/genética , Cefotaxima/farmacologia , Ácido Clavulânico/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Luciferases/antagonistas & inibidores , Luciferases/genética , Relação Estrutura-Atividade , Sulbactam/farmacologia , beta-Galactosidase/antagonistas & inibidores , beta-Galactosidase/genética , Inibidores de beta-Lactamases , beta-Lactamases/genéticaRESUMO
We report in vitro (cell-free) protein expression in a microfluidic device using passive pumping. The polystyrene device contains 192 microchannels, each of which is connected to two wells positioned in a 384-well microplate format. A larger droplet of an expression solution was placed at one well of each channel while a smaller droplet of a nutrient solution was at the other well. Protein expression took place in the larger droplet and we found the expression yield in the expression solution is enhanced due to the replenishment of the nutrient solution supplied by passive pumping via the channel. The pumping pressure was generated from the difference in the surface tension between two different sized droplets at the two wells. We demonstrated expression of luciferase in the device and the expression yield was measured using luminescence assay. Different experimental conditions were investigated to achieve maximum protein yield with the least amount of reagents. Protein expression yields were found to be dependent on the amount of the nutrient solution pumped, independent of the amount of the expression solution within the experimental conditions studied. A higher feeding frequency or delivery rate of the nutrient solution resulted in higher protein expression yield. The work demonstrated the feasibility of using the microchannel array for protein expression with the following advantages: (1) simultaneous production of the same protein with different conditions to optimize the expression process; (2) simultaneous production of different proteins for high-throughput protein expression with high yield; (3) low reagent cost due to the fact that it consumes 125-800 times less than the amount used in a protein expression instrument commercially available.
Assuntos
Microfluídica/instrumentação , Proteínas/metabolismo , Sistema Livre de Células , Desenho de EquipamentoRESUMO
We report the development of a microfluidic array device for continuous-exchange, cell-free protein synthesis. The advantages of protein expression in the microfluidic array include (1) the potential to achieve high-throughput protein expression, matching the throughput of gene discovery; (2) more than 2 orders of magnitude reduction in reagent consumption, decreasing the cost of protein synthesis; and (3) the possibility to integrate with detection for rapid protein analysis, eliminating the need to harvest proteins. The device consists of an array of units, and each unit can be used for production of an individual protein. The unit comprises a tray chamber for in vitro protein expression and a well chamber as a nutrient reservoir. The tray is nested in the well, and they are separated by a dialysis membrane and connected through a microfluidic connection that provides a means to supply nutrients and remove the reaction byproducts. The device is demonstrated by synthesis of green fluorescent protein, chloramphenicol acetyl-transferase, and luciferase. Protein expression in the device lasts 5-10 times longer and the production yield is 13-22 times higher than in a microcentrifuge tube. In addition, we studied the effects of the operation temperature and hydrostatic flow on the protein production yield.