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1.
Prep Biochem Biotechnol ; 49(8): 813-821, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31169457

RESUMO

Separation of biomass from culture media by centrifugation and then washing the biomass are mandatory steps in the fermentation process of recombinant Pichia pastoris expressed HBsAg intracellularly. Biomass has to be washed many times to eliminate the culture media residues thoroughly. In this study, we tried to develop the hydrocyclone as an alternative method for separation of biomass from fermentation culture, an attractive replacement for centrifugation processes. The advantages of using hydrocyclone in biomass separation could be summarized in its suitability for continuous separation and its low risk of contamination. To evaluate the performance of hydrocyclone, concentration ratio in underflow to feed stream, capacity, and centrifugal force by considering three parameters of pressure drop, concentration, and the type of hydrocyclone were investigated. Using three level factorial design a concentration ratio equation was developed, with the correlation coefficient R2 = 0.977 ensured the good fitness of the predicted data with the experimental results. In optimal conditions, maximum concentration ratio was 1.246, for flow rate 13.5 LPM and C-force equal to 1276.11 at maximum pressure drop (3 bar) and minimum concentration (0.5% w/w) in hydrocyclone 1. Herein, two different hydrocyclones with the cylindrical diameters of 19 mm and 21 mm were used for separating the yeast cells.


Assuntos
Centrifugação/instrumentação , Meios de Cultura/química , Antígenos de Superfície da Hepatite B/isolamento & purificação , Pichia/química , Técnicas de Cultura Celular por Lotes/instrumentação , Biomassa , Desenho de Equipamento , Fermentação , Pressão , Proteínas Recombinantes/isolamento & purificação
2.
Matern Child Nutr ; 15 Suppl 3: e12791, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31148399

RESUMO

Good nutrition during a child's early years lays a strong foundation of health for the rest of its life. Yet in India, there is widespread prevalence of undernourishment among children below 5 years of age. Within the Indian context, small millets have great potential as a healthy food to address this challenge by the virtue of their nutritional qualities. However, there are many problems with the current processing technology for small millets, whereas the use of value-added products was minimal. To address this, an assessment of existing small millet processing machinery was undertaken, and a double chamber centrifugal dehuller was developed, which had higher recovery of dehulled unpolished millets and met requirements at the village and enterprise levels. To demonstrate the health benefits of consuming value-added small millets, a study of supplementation of multi-millet health mix on the nutritional status of primary schoolchildren was conducted in Thondamuthur Block of Coimbatore District, India. Multi-millet health mix was formulated from kodo millet, little millet, foxtail millet, finger millet, and wheat with the inclusion of pulses. It contained 65.45-g carbohydrate, 11.46-g protein, 4.94-g fat, 4.94-g fibre, 4.07-mg iron, 112-mg calcium, 268.52-mg phosphorus, and 349 calories of energy per 100 g. The study indicated that there was a significant increase in height, weight, and haemoglobin level of the schoolchildren who regularly consumed the formulated multi-millet health mix. The improved huller and value-added food product developed can be feasible options for improving nutrition security and livelihoods through increased use of small millets.


Assuntos
Centrifugação/instrumentação , Suplementos Nutricionais , Desnutrição/prevenção & controle , Milhetes , Valor Nutritivo , Panicum , Criança , Pré-Escolar , Grão Comestível , Tecnologia de Alimentos/instrumentação , Humanos , Índia/epidemiologia , Estado Nutricional
3.
Biosens Bioelectron ; 141: 111466, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31254863

RESUMO

Since the emergence of the lab-on-a-chip technology in 1979, a variety of microfluidic devices have been developed and utilized for chemical and biological applications. Among the microfluidic devices, the centrifugal microfluidic device or lab-on-a-disc (LOAD) has advanced remarkably due to simple operation by the rotation, total integration, and high-throughput capability. Moreover, the centrifugal microdevices do not need complex tubing and pumping systems, which render them ideal for point-of-care testing (POCT) system. Owing to these characteristics, the centrifugal microdevices have been extensively used for bio-diagnostics. In particular, molecular diagnostics, which are regarded as an essential method for definite determination of the targets related with diseases, have been widely applied on the LOAD. In this review paper, we focus on the molecular diagnostics on the LOAD. The steps for the molecular diagnostics such as cell lysis, genome purification, gene amplification, amplicon detection, and data analysis can be performed individually or totally on the LOAD. Future directions of the LOAD in the fields of bio-diagnostics is to realize POCT for U-healthcare monitoring. In this context, the latest LOAD strategies for molecular diagnostics are summarized in this review paper, which would provide an insight for future POCT platform.


Assuntos
Dispositivos Lab-On-A-Chip , Ácidos Nucleicos/análise , Testes Imediatos , Reação em Cadeia da Polimerase/instrumentação , Animais , Centrifugação/instrumentação , Desenho de Equipamento , Humanos , Ácidos Nucleicos/genética
4.
PLoS Biol ; 17(5): e3000251, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31112539

RESUMO

The centrifuge is an essential tool for many aspects of research and medical diagnostics. However, conventional centrifuges are often inaccessible outside of standard laboratory settings, such as remote field sites, because they require a constant external power source and can be prohibitively costly in resource-limited settings and Science, technology, engineering, and mathematics (STEM)-focused programs. Here we present the 3D-Fuge, a 3D-printed hand-powered centrifuge, as a novel alternative to standard benchtop centrifuges. Based on the design principles of a paper-based centrifuge, this 3D-printed instrument increases the volume capacity to 2 mL and can reach hand-powered centrifugation speeds up to 6,000 rpm. The 3D-Fuge devices presented here are capable of centrifugation of a wide variety of different solutions such as spinning down samples for biomarker applications and performing nucleotide extractions as part of a portable molecular lab setup. We introduce the design and proof-of-principle trials that demonstrate the utility of low-cost 3D-printed centrifuges for use in remote field biology and educational settings.


Assuntos
Centrifugação/instrumentação , Biologia Molecular , Impressão Tridimensional/instrumentação , Genômica , Nanoporos , Nucleotídeos/isolamento & purificação , Proteínas/análise , Floresta Úmida , Manejo de Espécimes , Biologia Sintética
5.
Lab Chip ; 19(11): 1941-1952, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-30997461

RESUMO

This paper describes the development of an on-chip nucleic acid (NA) extraction assay from whole blood using a centrifugal microfluidic platform that allows for pneumatic actuation of liquids during rotation. The combination of pneumatic and centrifugal forces makes it possible to perform fluidic operations without the need for integrating active control elements on the microfluidic cartridge. The cartridge is fabricated from thermoplastic polymers (e.g., Zeonor 1060R) and features a simple design that is compatible with injection molding. In addition, the cartridge is interfaced with two external vials for off-chip storage of the blood sample and retrieval of the eluted NA solution, respectively. On-chip capture of NAs is performed using an embedded solid-phase extraction matrix composed of commercial glass microfiber filters (Whatman GF/D and GF/F). The yield of the automated, on-chip extraction protocol, determined by measuring absorbance at 260 nm, is comparable to some of the best manually operated kits (e.g., Qiagen QIAamp DNA Mini Kit) while providing low assay-to-assay variability due to the high level of control provided by the platform for each processing step. The A260/A280 and A260/A230 ratios of the absorbance spectra also reveal that protein contamination of the sample is negligible. The capability of the pneumatic platform to circulate air flux through the microfluidic conduit was used to dry leftover ethanol residues retained in the capture matrix during washing. This method, applied in combination with localized heating, proved effective for reducing ethanol contamination in eluted samples from ∼12% to 1% (v/v).


Assuntos
Métodos Analíticos de Preparação de Amostras/métodos , Centrifugação/instrumentação , Dispositivos Lab-On-A-Chip , Ácidos Nucleicos/sangue , Ácidos Nucleicos/isolamento & purificação , Automação , DNA Bacteriano/sangue , DNA Bacteriano/isolamento & purificação , Desenho de Equipamento , Escherichia coli O157/genética
6.
Lab Chip ; 19(10): 1728-1735, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31020298

RESUMO

The lab-on-a-disc is a powerful microfluidic platform that skillfully takes advantage of centrifugal force to controllably drive liquids with the assistance of passive or active valves. However, the passive valves are mainly triggered by the rotation speed and can be easily influenced by the surface chemistry of the channel, while the active valves usually require a complicated fabrication or actuation procedure. In this study, a novel active valve that can be easily triggered by an electromagnet was proposed and applied on the centrifugation platform. This valve, named the electromagnet-triggered pillar (ETP) valve, consisted of a metal pin and pressure sensitive adhesive (PSA) tape, and is closed until the pin is lifted up by an electromagnet to partially separate the PSA tape from the substrate. As a typical application, this valve is utilized to construct a centrifugal chip for mycotoxin detection. With four ETP valves in a unit, the sample and liquid reagents can be sequentially released into the reaction chamber that was spotted with mycotoxin conjugates to accomplish the whole immunoassay. Four mycotoxins (aflatoxin B1, ochratoxin A, T-2 toxin, and zearalenone) were simultaneously detected on this chip with limits of detection lower than the permissible limits set by the regulatory agencies of China, demonstrating the practicability of this easy-to-use active valve.


Assuntos
Centrifugação/instrumentação , Imunoensaio/instrumentação , Imunoensaio/métodos , Imãs , Técnicas Analíticas Microfluídicas , Micotoxinas/análise , Animais , Bovinos , Camundongos , Soroalbumina Bovina/química
7.
Lab Chip ; 19(9): 1657-1664, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30931470

RESUMO

Nucleic acid amplification methods are increasingly being used to detect trace quantities of DNA in samples for various diagnostic applications. However, quantifying the amount of DNA from such methods often requires time consuming purification, washing or labeling steps. Here, we report a novel microfluidic centrifugation assisted precipitation (µCAP) method for single-step DNA quantification. The method is based on formation of a visible precipitate, which can be quantified, when an intercalating dye (GelRed) is added to the DNA sample and centrifuged for a few seconds. We describe the mechanism leading to the precipitation phenomenon. We utilize centrifugal microfluidics to precisely control the formation of the visible and quantifiable mass. Using a standard CMOS sensor for imaging, we report a detection limit of 45 ng µl-1. Furthermore, using an integrated lab-on-DVD platform we recently developed, the detection limit is lowered to 10 ng µl-1, which is comparable to those of current commercially available instruments for DNA quantification. As a proof of principle, we demonstrate the quantification of LAMP products for a HIV-1B type genome containing plasmid on the lab-on-DVD platform. The simple DNA quantification system could facilitate advanced point of care molecular diagnostics.


Assuntos
Centrifugação/instrumentação , Precipitação Química , DNA/análise , DNA/isolamento & purificação , Dispositivos Lab-On-A-Chip , DNA/genética , Genoma Viral/genética , HIV-1/genética , Técnicas de Amplificação de Ácido Nucleico
8.
Forensic Sci Int ; 297: 204-216, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30831412

RESUMO

Diatom analysis is very effective for positive diagnosis of water inhalation in drowning. However, conventional strong acid diatom testing is laborious and potentially dangerous. We propose a simple, fast, and safe protocol using inexpensive reagents such as papain, SDS, and 5 N HCl for extracting diatoms from lung, kidney, and liver tissues. First, we determined optimal conditions for papain digestion using porcine tissues. Papain digestion was clearly superior to Proteinase K digestion. Next, for assessing the assay effectiveness in practical cases, the papain digestion protocol was applied to 80 tissue samples from 20 suspected drowning victims. Left and right lung tissues (1 g each) were digested in 15-mL conical centrifuge tubes. Kidney and liver tissues (10 g each) were extracted in 175-mL conical centrifuge bottles. Papain dissolved all organs sufficiently and permitted clear visualization of diatoms, although papain's solubilization activity was still inferior to strong acid digestion. The proposed enzymatic method requires only a low-speed centrifuge and water bath. Diatoms typically can be extracted from tissue samples within 3-5 h. The cost of protease is reduced some 6-fold by using papain in place of Proteinase K. Thus, the proposed method can be useful as a less-laborious, less-hazardous, and less-costly minimal test when the conventional strong acid digestion method is not performed due to personnel, equipment, budgetary limitation, or environmental and safety considerations.


Assuntos
Diatomáceas/isolamento & purificação , Afogamento/diagnóstico , Patologia Legal/métodos , Indicadores e Reagentes , Papaína , Animais , Cadáver , Centrifugação/instrumentação , Endopeptidase K , Humanos , Incubadoras , Rim/patologia , Fígado/patologia , Pulmão/patologia , Manejo de Espécimes/métodos , Suínos
9.
Int J Artif Organs ; 42(6): 291-298, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30854913

RESUMO

Flow rate estimation for ventricular assist devices without additional flow sensors can improve the quality of life of patients. In this article, a novel flow estimation method using the passively stabilized displacement of a magnetically levitated impeller is developed to achieve sufficient accuracy and precision of flow estimation for ventricular assist devices in a simple manner. The magnetically levitated impeller used is axially suspended by a magnetic bearing in a centrifugal blood pump that has been developed by our group. The radial displacement of the impeller, which is restricted by passive stability, can be correlated with the flow rate because the radial hydraulic force on the impeller varies according to the flow rate. To obtain the correlation with various blood viscosities, the relationships between the radial displacements of the magnetically levitated impeller and the pressure head-flow rate characteristics of the pump were determined simultaneously using aqueous solutions of glycerol with a potential blood viscosity range. The measurement results showed that accurate steady flow rates could be estimated with a coefficient of determination of approximately 0.97 and mean absolute error of approximately 0.22 L/min without fluid viscosity measurements by using the relationships between the impeller displacement and the flow rate. Moreover, a precision of approximately 0.01 (L/min)/µm was obtained owing to a strong estimation indicator signal provided by the large displacement of the passively stabilized impeller; thus, the proposed estimation method can help ensure sufficient accuracy and precision for ventricular assist devices in a simple manner, even if the blood viscosity is unknown.


Assuntos
Centrifugação , Coração Auxiliar/normas , Qualidade de Vida , Velocidade do Fluxo Sanguíneo , Centrifugação/instrumentação , Centrifugação/métodos , Desenho de Equipamento , Insuficiência Cardíaca/psicologia , Insuficiência Cardíaca/terapia , Humanos , Imãs
10.
J Chromatogr A ; 1596: 134-141, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-30862407

RESUMO

The partition efficiencies of three different coiled columns, conventional multilayer coiled column, eccentric coiled column and toroidal coiled column, were evaluated by the separation of 4-methylumbelliferyl sugar derivatives using the coil satellite centrifuge (CSC) with an organic-aqueous two-phase solvent system composed of ethyl acetate/1-butanol/water. The CSC apparatus was reinforced the planet axis to maintain the stable satellite motion, which was completed by combining the rotation of three axes including the sun axis (the angular velocity, ω1), the planet axis (ω2) and the satellite axis (ω3) under the relation at ω1 = ω2 + ω3. In the present study, four different rotation speed combination types were used for the separation at the ratio (ω1, ω2, ω3) = I. (300, 150, 150), II. (300, 100, 200), III. (300, 147, 153) and IV. (300, 200, 100 rpm) under different revolution speeds of ω1 = 300, 400 and 500 rpm. In the conventional multilayer coiled column with the upper mobile phase, the rotation speed combination type II yielded the best peak resolution while the rotation speed combination types III and IV had extremely low stationary phase retention even at higher revolution speeds. This inconvenience was eliminated by using the eccentric and the toroidal coiled column. The rotation speed combination type II for the eccentric coiled column and the type IV for the toroidal coiled column produced the best separation in both the upper and the lower mobile phases among four different rotation speed combination types. The overall results indicated that better peak resolution was obtained by the eccentric coiled column than by the toroidal coiled column except for the separation with the upper mobile phase at the low revolution speed.


Assuntos
Centrifugação/instrumentação , Técnicas de Química Analítica/métodos , 1-Butanol/química , Acetatos/química , Rotação , Solventes/química , Água/química
11.
Chemosphere ; 222: 671-678, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30735967

RESUMO

Measurement and reporting of concentrations of contaminants of emerging concern such as per- and polyfluoroalkyl substances (PFASs), including perfluorooctanoic acid (PFOA), is an integral part of most investigations. Occurrence of sorption losses of PFAS analytes onto particular laboratory-ware (e.g. glass containers) has been suggested in the published literature but has not been investigated in detail. We examined sorption losses from aqueous PFOA solutions in contact with different commonly-used materials in filter units and centrifuge tubes (glass and plastics). Sorption of PFOA onto different filter membrane types ranged from 21-79% indicating that filtration can introduce a major source of error in PFOA analysis; pre-treatment of filter membranes with phosphate or methanol solutions did not improve PFOA recovery. Substantial adsorption of PFOA was also observed on tubes made from polypropylene (PP), polystyrene (PS), polycarbonate (PC), and glass where losses observed were between 32-45%, 27-35%, 16-31% and 14-24%, respectively. Contrary to suggestions in the literature, our results indicated that the greatest sorption losses for PFOA occurred on PP, whereas losses on glass tubes were much lower. Variations in ionic strength and pH did not greatly influence PFOA recovery. When PFOA concentrations were increased, the percent recovery of PFOA increased, indicating that binding sites on tube-walls were saturable. This study draws attention towards analytical bias that can occur due to sorption losses during routine procedures, and highlights the importance of testing the suitability of chosen laboratory-ware for specific PFAS analytes of interest prior to experimental use.


Assuntos
Adsorção , Caprilatos/análise , Poluentes Ambientais/análise , Fluorcarbonetos/análise , Centrifugação/instrumentação , Filtração/instrumentação , Poluentes Químicos da Água/isolamento & purificação
12.
Talanta ; 194: 903-909, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30609623

RESUMO

A microfluidic SERS chip integrated with blood separation and in-situ detection was designed and fabricated for the rapid detection of clinical blood samples. Each functional unit in the microfluidic SERS chip consist of separation-decantation cavity based on centrifugal separation principle, mixing channels and SERS detection chamber built with integrated nano-Au on Ag film microstructure. The serum creatinine was selected as a typical sample to demonstrate the capability of microfluidic SERS chip. It was found that the creatinine SERS characteristic peaks at 678 cm-1 can be effectively identified and the detection limit could be as low as 4.42 × 10-3 µmol mL-1 in water. The blood samples were also tested in microfluidic SERS chip. The whole separation and test process could be completed within 2 min, which is a significant improvement in the field of creatinine detection. The whole blood of six cases clinical blood samples were also tested, and the results were consistent with the enzymatic results. The developed microfluidic SERS chip has advantages including reduction of the required quantity of blood sample, reusable and easy to operate. It is expected to provide a new method for rapid diagnostics.


Assuntos
Centrifugação/instrumentação , Dispositivos Lab-On-A-Chip , Testes Imediatos , Análise Espectral Raman/instrumentação , Desenho de Equipamento , Ouro/química , Humanos , Falência Renal Crônica/diagnóstico , Nanopartículas Metálicas/química , Prata/química , Fatores de Tempo
13.
Anaerobe ; 55: 61-66, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30315963

RESUMO

Clostridium difficile is a Gram-positive spore forming rod-shaped bacterium which causes mild to severe diarrhea. Spores play a key role in transmission of C. difficile in hospital environment. To investigate ability of spores to stay on fomites and to assess levels of contamination it is essential to prevent loss of spores collected for the analysis. Working with C. difficile spores we noticed a significant loss after vortexing of spore suspensions and investigated if it can be prevented by using a specific brand or type of microcentrifuge tubes. 7 types of microcentrifuge tubes from 3 manufacturers were tested. Spores of three types of C. difficile, NAP1/027, NAP4/014 and NAP7/078 (clinical isolates) were used. C. difficile was grown on Brucella Supplemented Agar for 9 days, spores were collected, washed and density of 3 suspensions was normalized to optical density (OD) 550 0.1 or 0.05. These suspensions (OD 0.1) were used in serial dilutions with 3 experimental conditions - pipetting, vortexing or vortexing in 3% albumin solution and in vortexing experiment when 150 µl were vortexed for 1 min at 1500 rpm per tube and loss of spores was measured by a decrease in dipicolinic acid (DPA) concentration measured by time-delayed terbium fluorescence. Inner surface of the tubes was visualized with microscopy to observe adhered spores. In serial dilution experiment, initial concentration of spores would be underestimated by up to 18X in case of vortexing for NAP1 strain and 9X for NAP4 strain. Presence of 3% albumin significantly decreases this effect but does not eliminate it completely. Comparison of 7 types of tubes shows that a single vortexing for 1 min of diluted spore suspension at concentrations of 1.8 × 107 spores per ml leads to a loss of up to 90% of spores in some tubes. Degree of spores' adhesion varied between brands and types of tubes and between tubes of the same type. In some brands there was a significant variability in adhesion between tubes from the same batch. Microscopy after vortexing shows a film of spores attached to the tube's wall. Adherence could be affected by the type of plastic, additives (plasticizers) used in manufacturing and quality of moulds (e.g."diamond polished"). To identify the most appropriate type of tubes for the experiment it is essential to test it beforehand as not every brand is suitable for this purpose. Using tubes with a high degree of adherence could significantly affect measurement of spores' concentration in serial dilutions, e.g. when quantifying spores production by a specific strain or when limits of detection are measured. Also, sensitivity of commercial tests for detection of C. difficile in clinical specimens can be decreased if an unsuitable type of plastic containers and tubes is used.


Assuntos
Centrifugação/instrumentação , Contenção de Riscos Biológicos , Segurança de Equipamentos , Técnicas Microbiológicas/instrumentação , Carga Bacteriana , Esporos Bacterianos/isolamento & purificação
14.
Tree Physiol ; 39(1): 156-165, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29788216

RESUMO

Cavitation resistance is a key trait for characterizing the drought adaption in plants and is usually presented in terms of vulnerability curves. Three principal techniques have been developed to produce vulnerability curves, but curves generated with centrifugation are reported to suffer from artifacts when applied to long-vesseled species. The main cause of this artifact is the issue of open vessels, resulting in a nano-particle effect that may seed premature embolism. We used two methods to test the potential mechanism behind the nano-particle effect in centrifuge-based vulnerability curves. A four-cuvette rotor system based on a traditional Cochard rotor was designed to inhibit effervescence while injecting water, but the recalcitrant vulnerability curves in Robinia could not be eliminated. There may be multiple sources, besides effervescence, of hypothetical nano-particles: they may arise from cut surfaces or they may be always present in the injected water, leading to the premature embolisms. To prevent the entry of the hypothetical nano-particles, water extraction curves in terms of PLV (percentage loss volume of extracted water from stems) vs tensions were constructed. The PLV curves of Robinia showed s-shaped characteristics after subtracting the first Weibull components from water extraction curves, which were not related to the water loss from vessels according to dye staining experiments. The differences between T50 (xylem tension at which 50% of hydraulic conductivity is lost) in mean PLV curve and T50 in percentage loss of conductivity curves determined by the four-cuvette rotor system and by the bench dehydration method were 3.9 MPa and 0.7 MPa, respectively. Hence, PLV curves may be a valid way to measure the cavitation resistance in long-vesseled species with centrifugation. Keeping bark intact in the process of measurement is recommended, otherwise it would increase evaporation from the entire system.


Assuntos
Aclimatação , Centrifugação/métodos , Nanopartículas , Doenças das Plantas , Robinia/fisiologia , Calibragem , Centrifugação/instrumentação , Resistência à Doença , Secas , Caules de Planta/fisiologia , Água
15.
Talanta ; 190: 134-139, 2018 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-30172489

RESUMO

Diabetes mellitus is a global endemic with a rapidly increasing prevalence in both developing and developed countries. Recently, hemoglobin A1c has been recommended by the American Diabetes Associations as a possible substitute for fasting blood glucose for the diagnosis of diabetes, because it is an indicator of long-term glycemic control. Also, centrifugal microfluidic systems have good potential for use in the point of care testing systems. In this study, a centrifugal microfluidic disc was designed and manufactured to measure hemoglobin A1c in whole blood using an immunoturbidimetry based method. Also, a new passive valve, named septum valve, was presented to precisely control the entry and exit of reagents. This design comprises three inputs for injection of reagents and a blood sample, three septum valves, a two-part mixing chamber and a chamber to measure the absorbance of the sample based on the immunoturbidimetry method. Fourteen blood samples were tested using the manufactured disc, and the results were very congruous with the clinical data. By using the designed centrifugal microfluidic disc, the hemoglobin A1c in whole blood was measured in 8 min, with a standard deviation of ±â€¯0.36% HbA1c.


Assuntos
Análise Química do Sangue/instrumentação , Centrifugação/instrumentação , Hemoglobina A Glicada/análise , Dispositivos Lab-On-A-Chip , Calibragem , Desenho de Equipamento , Humanos , Hidrodinâmica
16.
J Proteome Res ; 17(9): 2917-2924, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30114372

RESUMO

The success of shotgun proteomic analysis depends largely on how samples are prepared. Current approaches (such as those that are gel-, solution-, or filter-based), although being extensively employed in the field, are time-consuming and less effective with respect to the repetitive sample processing, recovery, and overall yield. As an alternative, the suspension trapping (S-Trap) filter has been commercially available very recently in the format of a single or 96-well filter plate. In contrast to the conventional filter-aided sample preparation (FASP) approach, which utilizes a molecular weight cut-off (MWCO) membrane as the filter and requires hours of processing before digestion-ready proteins can be obtained, the S-Trap employs a three-dimensional porous material as filter media and traps particulate protein suspensions with the subsequent depletion of interfering substances and in-filter digestion. Due to the large (submicron) pore size, each centrifugation cycle of the S-Trap filter only takes 1 min, which significantly reduces the total processing time from approximately 3 h by FASP to less than 15 min, suggesting an ultrafast sample-preparation approach for shotgun proteomics. Here, we comprehensively evaluate the performance of the individual S-Trap filter and 96-well filter plate in the context of global protein identification and quantitation using whole-cell lysate and clinically relevant sputum samples.


Assuntos
Filtração/métodos , Klebsiella pneumoniae/química , Proteômica/métodos , Manejo de Espécimes/métodos , Escarro/química , Tuberculose Pulmonar/metabolismo , Proteínas de Bactérias , Centrifugação/instrumentação , Centrifugação/métodos , Cromatografia Líquida/instrumentação , Etiópia , Filtração/instrumentação , Interações Hospedeiro-Patógeno , Humanos , Membranas Artificiais , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/patogenicidade , Proteólise , Proteômica/instrumentação , Espectrometria de Massas em Tandem/instrumentação , Tuberculose Pulmonar/microbiologia
17.
Soft Matter ; 14(26): 5356-5363, 2018 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-29781012

RESUMO

One of the common operations in sample preparation is to separate specific particles (e.g. target cells, embryos or microparticles) from non-target substances (e.g. bacteria) in a fluid and to wash them into clean buffers for further processing like detection (called solution exchange in this paper). For instance, solution exchange is widely needed in preparing fluidic samples for biosensing at the point-of-care and point-of-use, but still conducted via the use of cumbersome and time-consuming off-chip analyte washing and purification techniques. Existing small-scale and handheld active and passive devices for washing particles are often limited to very low throughputs or require external sources of energy. Here, we integrated Dean flow recirculation of two fluids in curved microchannels with selective inertial focusing of target particles to develop a microfluidic centrifuge device that can isolate specific particles (as surrogates for target analytes) from bacteria and wash them into a clean buffer at high throughput and efficiency. We could process micron-size particles at a flow rate of 1 mL min-1 and achieve throughputs higher than 104 particles per second. Our results reveal that the device is capable of singleplex solution exchange of 11 µm and 19 µm particles with efficiencies of 86 ± 2% and 93 ± 0.7%, respectively. A purity of 96 ± 2% was achieved in the duplex experiments where 11 µm particles were isolated from 4 µm particles. Application of our device in biological assays was shown by performing duplex experiments where 11 µm or 19 µm particles were isolated from an Escherichia coli bacterial suspension with purities of 91-98%. We envision that our technique will have applications in point-of-care devices for simultaneous purification and solution exchange of cells and embryos from smaller substances in high-volume suspensions at high throughput and efficiency.


Assuntos
Centrifugação/instrumentação , Escherichia coli K12/isolamento & purificação , Dispositivos Lab-On-A-Chip , Microesferas , Fatores de Tempo
18.
PLoS One ; 13(4): e0195907, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29659624

RESUMO

The centrifuge is among the oldest and most widely used pieces of laboratory equipment, with significant applications that include clinical diagnostics and biomedical research. A major limitation of laboratory centrifuges is their "black box" nature, limiting sample observation to before and after centrifugation. Thus, optimized protocols require significant trial and error, while unoptimized protocols waste time by centrifuging longer than necessary or material due to incomplete sedimentation. Here, we developed an instrumented centrifuge tube receptacle compatible with several commercial benchtop centrifuges that can provide real-time sample analysis during centrifugation. We demonstrated the system by monitoring cell separations during centrifugation for different spin speeds, concentrations, buffers, cell types, and temperatures. We show that the collected data are valuable for analytical purposes (e.g. quality control), or as feedback to the user or the instrument. For the latter, we verified an adaptation where complete sedimentation turned off the centrifuge and notified the user by a text message. Our system adds new functionality to existing laboratory centrifuges, saving users time and providing useful feedback. This add-on potentially enables new analytical applications for an instrument that has remained largely unchanged for decades.


Assuntos
Centrifugação/instrumentação , Centrifugação/métodos , Separação Celular/métodos , Desenho de Equipamento , Laboratórios , Impressão Tridimensional
19.
Biochem Med (Zagreb) ; 28(2): 020902, 2018 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-29666561

RESUMO

Introduction: Obtaining suitable results unaffected by pre- or postanalytical phases is pivotal for clinical chemistry service. We aimed comparison and stability of nine biochemical analytes after centrifugation using Barricor™ plasma tubes with mechanical separator vs standard Vacutainer® lithium heparin tubes. Materials and methods: We collected samples on six healthy volunteers and nine patients from intensive care units into 6 mL plastic Vacutainer® lithium heparin tubes and 5.5 mL plastic Barricor™ plasma tubes. All tubes were centrifuged within 30 minutes after venipuncture. First, we compared results of nine biochemical analytes from lithium heparin tubes with Barricor™ tubes for each analyte using Passing-Bablok and Bland-Altman analyses. Second, we calculated the difference of analyte concentrations between baseline and time intervals in tubes stored at + 4 °C. Based on the total change limit we calculated the maximum allowable concentrations percentage change from baseline. Results: The majority of correlation coefficients were close to 0.99 indicating good correlation in the working range. Bland-Altman analyses showed an acceptable concordance for all analytes. In consequence, the Barricor™ tube might be an alternative to regular lithium heparin tube. Stability with this new generation tube is improved for eight analytes (except for aspartate aminotransferase) in comparison with regular lithium heparin tubes. Conclusions: By using Barricor™ tubes and prompt centrifugation, supplemental analysis or re-analysis for eight analytes including alanine aminotransferase, alkaline phosphatase, C-reactive protein, high sensitivity troponin T, lactate dehydrogenase, NT-pro BNP, potassium and sodium could be performed within 72 h of specimen collection.


Assuntos
Coleta de Amostras Sanguíneas/instrumentação , Centrifugação/instrumentação , Alanina Transaminase/sangue , Fosfatase Alcalina/sangue , Anticoagulantes/química , Biomarcadores/sangue , Coleta de Amostras Sanguíneas/normas , Proteína C-Reativa/metabolismo , Estudos de Casos e Controles , Cátions Monovalentes , Estado Terminal , Heparina/química , Humanos , Unidades de Terapia Intensiva , L-Lactato Desidrogenase/sangue , Lítio/química , Peptídeo Natriurético Encefálico/sangue , Fragmentos de Peptídeos/sangue , Potássio/sangue , Sódio/sangue , Troponina T/sangue
20.
Lab Chip ; 18(10): 1452-1460, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29664087

RESUMO

In almost any branch of chemistry or life sciences, it is often necessary to study the interaction between different components in a system by varying their respective concentrations in a systematic manner. Currently, many procedures for generating a series of samples of different solute concentration levels are still done manually by dilution. To address this issue, we present herein a highly automated linear concentration gradient generator based on centrifugal microfluidics. The operation of this device is based on the use of multi-layered microfluidics in which individual fluidic samples to be mixed together are stored and metered in their respective layers before finally being transferred to a mixing chamber. To demonstrate the operation of this scheme, we have used the device to conduct antimicrobial susceptibility testing (AST). Firstly, DI water, ampicillin solution and E. coli suspension were loaded into the chambers in different layers. As the device went through several rounds of spinning at different speeds, a series of metered dosages of ampicillin along a linear concentration gradient were introduced to the mixing chamber and mixed with E. coli automatically. By monitoring the spectral absorbance of the suspensions, we were able to establish the minimum inhibitory concentration (MIC) value of ampicillin against E. coli. The process took about 3 hours to complete, and the experimental results showed a strong correlation with those obtained with the standard CLSI broth dilution method. Clearly, the platform is useful for a wide range of applications such as drug discovery and personalised medicine, where concentration gradients are of concern.


Assuntos
Centrifugação/instrumentação , Testes de Sensibilidade Microbiana/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Ampicilina/farmacologia , Desenho de Equipamento , Escherichia coli/efeitos dos fármacos
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