RESUMO
We present a system efficiency analysis of a monolithic integrated organic optoelectronic unit for the detection of fluorescence labeled single-stranded DNA (ssDNA) for veterinary disease testing. The side-by-side integration of an organic light emitting diode (OLED) and an organic photodetector (OPD) with 0.5 mm by 0.5 mm device sizes has the potential to enable compact and low-cost fluorescence point-of-care (POC) devices for decentral multiplex biomedical testing. Here, we used two 6-FAM and BHQ1 labeled complementary ssDNA strands to form the Förster resonance transfer (FRET) upon the hybridization of the DNA. In this work we successfully show ssDNA hybridization sensing with samples diluted in TE buffer and investigate the detection of covalently bound 6-FAM-ssDNA on a glass surface for multiplex biomarker measurements.
RESUMO
A novel innovative approach towards a marketable lab-on-chip system for point-of-care in vitro diagnostics is reported. In a consortium of seven Fraunhofer Institutes a lab-on-chip system called "Fraunhofer ivD-platform" has been established which opens up the possibility for an on-site analysis at low costs. The system features a high degree of modularity and integration. Modularity allows the adaption of common and established assay types of various formats. Integration lets the system move from the laboratory to the point-of-need. By making use of the microarray format the lab-on-chip system also addresses new trends in biomedicine. Research topics such as personalized medicine or companion diagnostics show that multiparameter analyses are an added value for diagnostics, therapy as well as therapy control. These goals are addressed with a low-cost and self-contained cartridge, since reagents, microfluidic actuators and various sensors are integrated within the cartridge. In combination with a fully automated instrumentation (read-out and processing unit) a diagnostic assay can be performed in about 15 min. Via a user-friendly interface the read-out unit itself performs the assay protocol, data acquisition and data analysis. So far, example assays for nucleic acids (detection of different pathogens) and protein markers (such as CRP and PSA) have been established using an electrochemical read-out based on redoxcycling or an optical read-out based on total internal reflectance fluorescence (TIRF). It could be shown that the assay performance within the cartridge is similar to that found for the same assay in a microtiter plate. Furthermore, recent developments are the integration of sample preparation and polymerase chain reaction (PCR) on-chip. Hence, the instrument is capable of providing heating-and-cooling cycles necessary for DNA-amplification. In addition to scientific aspects also the production of such a lab-on-chip system was part of the development since this heavily affects the success of a later market launch. In summary, the Fraunhofer ivD-platform covers the whole value chain ranging from microfluidics, material and polymer sciences, assay and sensor development to the production and assembly design. In this consortium the gap between diagnostic needs and available technologies can be closed.
Assuntos
DNA Bacteriano/isolamento & purificação , DNA Fúngico/isolamento & purificação , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , RNA Ribossômico 18S/isolamento & purificação , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Desenho de Equipamento , Dispositivos Lab-On-A-Chip , Microfluídica/instrumentação , Microfluídica/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sistemas Automatizados de Assistência Junto ao LeitoRESUMO
The importance of early diagnosis devices increased continuously in the last two decades and plays an important role in medical care. Early stage diagnosis of e.g. ovarian cancer, HCV-infection or HIV-infection increased the survival rate of patients significantly. In parallel there is a trend leaving centralized diagnostic laboratories in order to get closer to the patient to perform analysis of even complex parameters in the field. This often saves time, increases the prognosis of the patient significantly and is cheaper in many cases. In this study we employ a rapid and cost-effective detection system based on electrical biochip technology for decentralized detection of anti-HCV Core immunoglobulins (HCV antibodies). In this system the qualitative and quantitative detection of virus-specific antibodies is done by an ELISA directly on a gold electrode array utilizing HCV Core as capture antigen. The biochip allows antibody detection within 20 min. Signal amplification was done by enzyme labelling and by "Single Electrode Redox Cycling". This method enhances current signals up to 40-fold in comparison to simple oxidation. The sensitivity of this approach is therefore comparable to a standard microtiter plate based ELISA with a 9-fold saving of assay time. This biochip system allows serum or whole blood analysis with no signal loss or increasing background caused by the red blood cells. Fields of application can be hospital emergency units where only single detections have to be conducted in a quick manner or by the general practitioner.
Assuntos
Técnicas Biossensoriais/instrumentação , Condutometria/instrumentação , Ensaio de Imunoadsorção Enzimática/instrumentação , Anticorpos Anti-Hepatite C/sangue , Imunoensaio/instrumentação , Imunoglobulinas/sangue , Desenho de Equipamento , Análise de Falha de Equipamento , Anticorpos Anti-Hepatite C/imunologia , Humanos , Imunoglobulinas/imunologiaRESUMO
A fully electrical array for voltammetric detection of redox molecules produced by enzyme-labeled affinity binding complexes is shown. The electronic detection is based on ultramicroelectrode arrays manufactured in silicon technology. The 200-microm circular array positions have 800-nm-wide interdigitated gold ultramicroelectrodes embedded in silicon dioxide. Immobilization of oligonucleotide capture probes onto the gold electrodes surfaces is accomplished via thiol-gold self-assembling. Spatial separation of probes at different array positions is controlled by polymeric rings around each array position. The affinity bound complexes are labeled with alkaline phosphatase, which converts the electrochemically inactive substrate 4-aminophenyl phosphate into the active 4-hydroxyaniline (HA). The nanoscaled electrodes are used to perform a sensitive detection of enzyme activity by signal enhancing redox recycling of HA resulting in local and position-specific current signals. Multiplexing and serial readout is realized using a CMOS ASIC module and a computer-controlled multichannel potentiostat. The principle of the silicon-based electrical biochip array is shown for different experimental setups and for the detection of virus DNA in real unpurified multiplex PCR samples. The fast and quantitative electronic multicomponent analysis for all kinds of affinity assays is robust and particle tolerant.
Assuntos
DNA Viral/análise , Microeletrodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , DNA Viral/química , Eletroquímica , Ensaio de Imunoadsorção Enzimática , Ouro/química , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Oxirredução , Reação em Cadeia da Polimerase , Silício/química , Propriedades de SuperfícieRESUMO
Based on electrical biochips made in Si-technology cost effective portable devices have been constructed for field applications and point of care diagnosis. These miniaturized amperometric biosensor devices enable the evaluation of biomolecular interactions by measuring the redox recycling of ELISA products, as well as the electrical monitoring of metabolites. The highly sensitive redox recycling is facilitated by interdigitated ultramicroelectrodes of high spatial resolution. The application of these electrical biochips as DNA microarrays for the molecular diagnosis of viral infections demonstrates the measurement procedure. Self-assembling of capture oligonucleotides via thiol-gold coupling has been used to construct the DNA interface on-chip. Another application for this electrical detection principle is continuous measuring with bead-based biosensors. Here, paramagnetic nanoparticles are used as carriers of the bioanalytical interface in ELISA format. A Si-micromachined glucose sensor for continuous monitoring in interstitial fluid ex vivo shows the flexibility of the electrical platform. Here the novel approach is a pore membrane in micrometer-dimensions acting as a diffusion barrier. The electrochemical detection takes place in a cavity containing glucose oxidase and a Pt-electrode surface. The common hydrogen peroxide detection, together with Si technology, enable precise differential measurements using a second cavity.