RESUMEN
A conceptually new technique for fast DNA detection has been developed. Here, we report a fast and sensitive online fluorescence resonance energy transfer (FRET) detection technique for label-free target DNA. This method is based on changes in the FRET signal resulting from the sequence-specific hybridization between two fluorescently labelled nucleic acid probes and target DNA in a PDMS microfluidic channel. Confocal laser-induced microscopy has been used for the detection of fluorescence signal changes. In the present study, DNA hybridizations could be detected without PCR amplification because the sensitivity of confocal laser-induced fluorescence detection is very high. Two probe DNA oligomers (5'-CTGAT TAGAG AGAGAA-TAMRA-3' and 5'-TET-ATGTC TGAGC TGCAGG-3') and target DNA (3'-GACTA ATCTC TCTCT TACAG GCACT ACAGA CTCGA CGTCC-5') were introduced into the channel by a microsyringe pump, and they were efficiently mixed by passing through the alligator teeth-shaped PDMS microfluidic channel. Here, the nucleic acid probes were terminally labelled with the fluorescent dyes, tetrafluororescein (TET) and tetramethyl-6-carboxyrhodamine (TAMRA), respectively. According to our confocal fluorescence measurements, the limit of detection of the target DNA is estimated to be 1.0 x 10(-6) to 1.0 x 10(-7)M. Our result demonstrates that this analytical technique is a promising diagnostic tool that can be applied to the real-time analysis of DNA targets in the solution phase.
Asunto(s)
Sondas de ADN/química , Sondas de ADN/genética , ADN/análisis , ADN/genética , Hibridación Fluorescente in Situ/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Diseño de Equipo , Análisis de Falla de Equipo , Hibridación Fluorescente in Situ/métodos , Coloración y Etiquetado/métodosRESUMEN
Confocal fluorescence microscopy (CFM) and confocal Raman microscopy (CRM) have been applied to monitor the laminar flow mixing behavior in a poly(dimethylsiloxane) (PDMS) microfluidic channel. Two passive PDMS micromixing devices were fabricated for this purpose: a two-dimensional round-wave channel and a three-dimensional serpentine channel. The microscale laminar flow mixing of ethanol and isopropanol was evaluated using the CFM and CRM at various flow rates. The mixing behavior of confluent streams in the microchannel was assessed by determining the degree of color change in Rhodamine 6G dye on mixing using the CFM. However, it was also possible to quantitatively evaluate the mixing process without employing a fluorescence label using the CRM. The results show a strong potential for CRM as a highly sensitive detection tool to measure fundamental fluid mixing processes and to provide detailed information on chemical changes of non-fluorescent reaction mixtures in a PDMS microfluidic channel.
Asunto(s)
Dimetilpolisiloxanos , Microquímica/métodos , Microfluídica/instrumentación , Microscopía Confocal/instrumentación , Siliconas , Espectrometría Raman/instrumentación , 2-Propanol/análisis , 2-Propanol/química , Etanol/análisis , Etanol/química , Microfluídica/métodos , Microscopía Confocal/métodos , Espectrometría Raman/métodosRESUMEN
The immobilization of glucose oxidase (GOx), using self assembled monolayers (SAMs) on gold surfaces, was investigated by grazing angle FT-IR spectroscopy, surface plasmon resonance (SPR) spectroscopy, and atomic force microscopy (AFM) in conjunction with confocal laser scanning microscopy (CLSM). To find an optimum condition for the maximum GOx loading density on gold surfaces, different cleaning protocols were examined. The loading density of GOx on surfaces was investigated by AFM and CLSM. In particular, CLSM was more effective for identifying the GOx density than AFM, since its scanning speed is much faster and it covers a larger area. Based on CLSM images of the GOx immobilized on the surfaces, it was concluded that the pre-cleaning process of gold substrates using different solvents, such as acetone, ethanol and 2-propanol, is very important for enhancing the GOx loading density. This result enables us to investigate an effective fabrication process in fabricating biosensors.
Asunto(s)
Técnicas Biosensibles/instrumentación , Enzimas Inmovilizadas/química , Glucosa Oxidasa/química , Oro , Técnicas Biosensibles/métodos , Microscopía de Fuerza Atómica , Microscopía Confocal , Espectroscopía Infrarroja por Transformada de Fourier , Resonancia por Plasmón de Superficie , Propiedades de SuperficieRESUMEN
Here we report the development of a programmable and fully automatic gold array-embedded gradient microfluidic chip that integrates a gradient microfluidic device with gold-patterned microarray wells. This device provides a convenient and reproducible surface-enhanced Raman scattering (SERS)-based immunoassay platform for cancer biomarkers. We used hollow gold nanospheres (HGNs) as SERS agents because of their highly sensitive and reproducible characteristics. The utility of this platform was demonstrated by the quantitative immunoassay of alpha-fetoprotein (AFP) model protein marker. Our proposed SERS-based immunoassay platform has many advantages over other previously reported SERS immunoassay methods. The tedious manual dilution process of repetitive pipetting and inaccurate dilution is eliminated with this process because various concentrations of biomarker are automatically generated by microfluidic gradient generators with N cascade-mixing stages. The total assay time from serial dilution to SERS detection takes less than 60 min because all of the experimental conditions for the formation and detection of immunocomplexes can be automatically controlled inside the exquisitely designed microfluidic channel. Thus, this novel SERS-based microfluidic assay technique is expected to be a powerful clinical tool for fast and sensitive cancer marker detection.
Asunto(s)
Oro/química , Inmunoensayo , Técnicas Analíticas Microfluídicas/instrumentación , Anticuerpos Inmovilizados/química , Anticuerpos Inmovilizados/inmunología , Humanos , Técnicas Analíticas Microfluídicas/métodos , Microscopía Fluorescente , Nanoestructuras/química , Análisis por Matrices de Proteínas , Espectrometría Raman , alfa-Fetoproteínas/análisisRESUMEN
This paper reports a highly reproducible immunoassay of cancer markers using surface-enhanced Raman scattering (SERS) imaging. SERS is a highly sensitive detection method but it is limited in its ability to achieve reproducible signal enhancement because of the difficulty with precisely controlling the uniform distribution of hot junctions. Consequently, inconsistent enhancement prevents the wide exploitation of SERS detection as a bio-detection tool for quantitative analysis. To resolve this problem, we explored the use of a SERS imaging-based immunoassay. For this purpose, Raman reporter-labeled hollow gold nanospheres (HGNs), were manufactured and antibodies were immobilized onto their surfaces for targeting specific antigens. After the formation of sandwich immunocomplexes using these functional HGNs on the surfaces of gold patterned wells, the SERS mapping images were measured. For target protein markers, 12×9 pixels were imaged using a Raman mapping technique in the 0-10(-4) g/mL concentration range, and the SERS signals for 66 pixels were averaged. Here, the SERS imaging-based assay shows much better correlations between concentration and intensity than does the conventional point-based assay. The limits of detection were determined to be 0.1 pg/mL and 1.0 pg/mL for angiogenin (ANG) and alpha-fetoprotein (AFP), respectively. This detection sensitivity is increased by three or four orders of magnitude over that of conventional ELISA method. The detectable dynamic range for SERS imaging (10(-4)-10(-12) g/mL) is also much wider than that for ELISA (10(-6)-10(-9) g/mL).
Asunto(s)
Biomarcadores de Tumor/análisis , Técnicas Biosensibles/instrumentación , Oro/química , Inmunoensayo/instrumentación , Proteínas de Neoplasias/análisis , Análisis por Matrices de Proteínas/instrumentación , Espectrometría Raman/instrumentación , Diseño de Equipo , Análisis de Falla de Equipo , Sensibilidad y EspecificidadRESUMEN
Antibody-conjugated hollow gold nanospheres (HGNs) have been used for the SERS imaging of HER2 cancer markers overexpressed in single MCF7 cells. SERS mapping images show that HGNs have much better homogeneous scattering properties than silver nanoparticles. The results demonstrate the potential feasibility of HGNs as highly sensitive and homogeneous sensing probes for biological imaging of cancer markers in live cells.
Asunto(s)
Anticuerpos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Oro , Nanoestructuras , Receptor ErbB-2/metabolismo , Espectrometría Raman/métodos , Biomarcadores de Tumor/inmunología , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/inmunología , Línea Celular Tumoral , Medios de Contraste/química , Oro/química , Humanos , Aumento de la Imagen/métodos , Microscopía/métodos , Nanoestructuras/química , Nanoestructuras/ultraestructuraRESUMEN
We have developed a sensitive, one-step, homogeneous open sandwich fluoroimmunoassay (OsFIA) based on fluorescence resonance energy transfer (FRET) and luminescent semiconductor quantum dots (QDs). In this FRET assay, estrogen receptor beta (ER-beta) antigen was incubated with QD-labeled anti-ER-beta monoclonal antibody and Alexa Fluor (AF)-labeled anti-ER polyclonal antibody for 30 min, followed by FRET measurement. The dye separation distance was estimated between 80 and 90 A. The current method is rapid, simple, and highly sensitive, and it did not require the bound/free reagent separation steps and solid-phase carriers. A concentration as low as 0.05 nM (2.65 ng/ml) receptor was detected with linearity. In addition, the assay was performed with commercial antibodies. This assay provides a convenient alternative to conventional, laborious sandwich immunoassays.