RESUMO
BACKGROUND: Miniaturization has not only driven microelectronics and generated new unforeseen options but has also dramatically changed sensors and analytics. DEVELOPMENTS: The Lab on a Chip (LOC) technology enables laboratory processes to run fully automated in canals in the micrometre range. The biggest challenge for LOC is to keep production costs low despite miniaturization and application-specific design. If this is achieved medical laboratory analyses can usually be carried out faster and with less hands on time. This explains why LOCs are already integrated into many laboratory instruments and why point-of-care testing (POCT) can no longer be imagined without it. New markers, such as in liquid biopsies and measurement techniques, such as Raman spectroscopy and mass spectroscopy, create further potentials that will enable faster and more specific laboratory analyses to be made using LOC technology. CONCLUSION: The LOC technology has the potential of changing the medical practice especially in cases when the central laboratory is not available or is unable to provide results fast enough.
Assuntos
Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Testes Imediatos , Telemedicina , HumanosRESUMO
An ultrafast microfluidic PCR module (30 PCR cycles in 6 min) based on the oscillating fluid plug concept was developed. A robust amplification of native genomic DNA from whole blood samples could be achieved at operational conditions established from systematic investigations of key parameters including heat transfer and in particular flow velocities. Experimental data were augmented with results from computational fluid dynamics simulations. The reproducibility of the current system was substantially improved compared to previous concepts by integration of a closed reservoir instead of utilizing a vented channel end at ambient pressure rendering the devised module suitable for integration into complex sample-to-answer analysis platforms such as point-of-care applications.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Actinas/genética , Simulação por Computador , DNA/sangue , DNA/química , Desenho de Equipamento , Humanos , Masculino , Sistemas Automatizados de Assistência Junto ao Leito , Reprodutibilidade dos Testes , TemperaturaRESUMO
During the developmental cycle of lab-on-a-chip devices, various microstructuring techniques are required. While in the designing and assay implementation phase direct structuring or so-called rapid-prototyping methods such as milling or laser ablation are applied, replication methods like hot embossing or injection moulding are favourable for large quantity manufacturing. This work investigated the applicability of rapid-prototyping techniques for thermoplastic chip development in general, and the reproducibility of performances in dependency of the structuring technique. A previously published chip for prenatal diagnosis that preconcentrates DNA via electrokinetic trapping and field-amplified-sample-stacking and afterwards separates it in CGE was chosen as a model. The impact of structuring, sealing, and the integration of membranes on the mobility of the EOF, DNA preconcentration, and DNA separation was studied. Structuring methods were found to significantly change the location where preconcentration of DNA occurs. However, effects on the mobility of the EOF and the separation quality of DNA were not observed. Exchange of the membrane has no effect on the chip performance, whereas the sealing method impairs the separation of DNA within the chip. The overall assay performance is not significantly influenced by different structuring methods; thus, the application of rapid-prototyping methods during a chip development cycle is well justified.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Plásticos/química , DNA/análise , Eletro-Osmose/instrumentação , Desenho de Equipamento , Membranas Artificiais , Cimento de Policarboxilato , Poliésteres , Diagnóstico Pré-Natal , Reprodutibilidade dos TestesRESUMO
Stopped-flow technology is frequently used to monitor rapid (bio)chemical reactions with high temporal resolution, e.g., in dynamic investigations of enzyme reactions, protein interactions, or molecular transport mechanisms. However, conventional stopped-flow devices are often overly complex, voluminous, or costly. Moreover, excessive amounts of sample are often wasted owing to inefficient designs. To address these shortcomings, we propose a stopped-flow system based on microfluidic design principles. Our simple and cost-efficient approach offers distinct advantages over existing technology. In particular, the use of injection-molded disposable microfluidic chips minimizes required sample volumes and associated costs, simplifies handling, and prevents adverse cross-contamination effects. The cost of the system developed is reduced by an order of magnitude compared with the cost of commercial systems. The system contains a high-precision valve system for fluid control and features automated data acquisition capability with high temporal resolution. Analyses with two well-established reaction kinetics yielded a dead time of approximately 8-9 ms.
Assuntos
Análise Custo-Benefício , Técnicas Analíticas Microfluídicas/economia , Técnicas Analíticas Microfluídicas/instrumentação , Polimetil Metacrilato/químicaRESUMO
In electrokinetic trapping (EKT), the electroosmotic velocity of a buffer solution in one area of a microfluidic device opposes the electrophoretic velocity of the analyte in a second area, resulting in transport of DNA to a location where the electrophoretic and electroosmotic velocities are equal and opposite and DNA concentrates at charged nanochannels. The method does not require an optical plug localization, a considerable advantage as compared to preconcentration techniques previously presented. In the work reported here, the trapping process is preceded by a field-amplification in the sample reservoir to reduce trapping time, as field-amplified EKT is shown to be an effective technique to preconcentrate samples from larger volumes. A theoretical model explaining the principle of field-amplified EKT considers different ionic strengths and cross-sectional areas in the microchip segments. The model is supported by experimental data using nucleic acids and fluorescein as sample analytes. An incorporated poly(ethylene terephthalate) (PET) membrane provides anion exclusion due to a negatively charged surface. A fluidic counter flow supports the trapping process in 100 nm pores due to anion exclusion. An analysis of Joule heating gives evidence that temperature gradient focusing effects are negligible and charge exclusion is responsible for trapping. The theoretical model developed and experimentally demonstrated can be exploited for the preconcentration of cell free fetal DNA circulating in maternal plasma and other rare nucleic acid species present in large sample volumes.
Assuntos
Métodos Analíticos de Preparação de Amostras/instrumentação , DNA/isolamento & purificação , Membranas Artificiais , Polietilenotereftalatos/química , Sistema Livre de Células , DNA/análise , DNA/sangue , DNA/química , Condutividade Elétrica , Eletroforese , Fluoresceína/química , Fluoresceína/isolamento & purificação , Técnicas Analíticas Microfluídicas , Modelos Químicos , Neoplasias/diagnóstico , Porosidade , TemperaturaRESUMO
BACKGROUND: Routine prenatal diagnosis of chromosomal anomalies is based on invasive procedures, which carry a risk of approximately 1%-2% for loss of pregnancy. An alternative to these inherently invasive techniques is to isolate fetal DNA circulating in the pregnant mother's plasma. Free fetal DNA circulates in maternal plasma primarily as fragments of lengths <500 bp, with a majority being <300 bp. Separating these fragments by size facilitates an increase in the ratio of fetal to maternal DNA. METHODS: We describe our development of a microsystem for the enrichment and isolation of cell-free fetal DNA from maternal plasma. The first step involves a high-volume extraction from large samples of maternal plasma. The resulting 80-microL eluate is introduced into a polymeric microsystem within which DNA is trapped and preconcentrated. This step is followed by a transient isotachophoresis step in which the sample stacks within a neighboring channel for subsequent size separation and is recovered via an outlet at the end of the channel. RESULTS: Recovered fractions of fetal DNA were concentrated 4-8 times over those in preconcentration samples. With plasma samples from pregnant women, we detected the fetal SRY gene (sex determining region Y) exclusively in the fragment fraction of <500 bp, whereas a LEP gene (leptin) fragment was detected in both the shorter and longer recovery fractions. CONCLUSIONS: The microdevice we have described has the potential to open new perspectives in noninvasive prenatal diagnosis by facilitating the isolation of fetal DNA from maternal plasma in an integrated, inexpensive, and easy-to-use microsystem.
Assuntos
DNA/isolamento & purificação , Feto , DNA/sangue , Feminino , Humanos , Dispositivos Lab-On-A-Chip , Membranas Artificiais , Procedimentos Analíticos em Microchip/métodos , Reação em Cadeia da Polimerase , Polimetil Metacrilato , Gravidez , Diagnóstico Pré-Natal/métodosRESUMO
In this paper, we describe the development of an automated sample preparation procedure for etiological agents of community-acquired lower respiratory tract infections (CA-LRTI). The consecutive assay steps, including sample re-suspension, pre-treatment, lysis, nucleic acid purification, and concentration, were integrated into a microfluidic lab-on-a-chip (LOC) cassette that is operated hands-free by a demonstrator setup, providing fluidic and valve actuation. The performance of the assay was evaluated on viral and Gram-positive and Gram-negative bacterial broth cultures previously sampled using a nasopharyngeal swab. Sample preparation on the microfluidic cassette resulted in higher or similar concentrations of pure bacterial DNA or viral RNA compared to manual benchtop experiments. The miniaturization and integration of the complete sample preparation procedure, to extract purified nucleic acids from real samples of CA-LRTI pathogens to, and above, lab quality and efficiency, represent important steps towards its application in a point-of-care test (POCT) for rapid diagnosis of CA-LRTI.
Assuntos
Infecções Comunitárias Adquiridas/microbiologia , Infecções Comunitárias Adquiridas/virologia , DNA Bacteriano/isolamento & purificação , Técnicas Analíticas Microfluídicas/métodos , RNA Viral/isolamento & purificação , Infecções Respiratórias/microbiologia , Infecções Respiratórias/virologia , Métodos Analíticos de Preparação de Amostras , Automação , Bactérias/genética , Fenômenos Fisiológicos Bacterianos , DNA Bacteriano/análise , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/fisiologia , Técnicas Analíticas Microfluídicas/instrumentação , RNA Viral/análiseRESUMO
A microsystem integrating electrochemical detection for the simultaneous detection of protein markers of breast cancer is reported. The microfluidic platform was realized by high precision milling of polycarbonate sheets and features two well distinguishable sections: a detection zone incorporating the electrode arrays and the fluid storage part. The detection area is divided into separate microfluidic chambers addressing selected electrodes for the measurement of samples and calibrators. The fluidic storage part of the platform consists of five reservoirs to store the reagents and sample, which are interfaced by septa. These reservoirs have the appropriate volume to run a single assay per cartridge and are manually filled. The liquids from the reservoirs are actuated by applying a positive air pressure (i.e.via a programmable syringe pump) through the septa and are driven to the detection zone via two turning valves. The application of the realised platform in the individual and simultaneous electrochemical detection of proteic cancer markers with very low detection limits are demonstrated. The microsystem has also been validated using real patient serum samples and excellent correlation with ELISA results obtained.