RESUMO
DNA sequencing by microchip capillary electrophoresis (CE) enables cheap, high-speed analysis of low reagent volumes. One of its potential applications is the identification of genomic deletions or insertions associated with genetic illnesses. Detecting single base-pair insertions or deletions from DNA fragments in the diagnostically relevant size range of 150-1000 base-pairs requires a variance of σ2 < 10-3. In a microfluidic chip post-processed by femtosecond-laser writing of an optical waveguide we CE-separated 12 blue-labeled and 23 red-labeled DNA fragments in size. Each set was excited by either of two lasers power-modulated at different frequencies, their fluorescence detected by a photomultiplier, and blue and red signals distinguished by Fourier analysis. We tested different calibration strategies. Choice of the fluorescent label as well as the applied fit function strongly influence the obtained variance, whereas fluctuations between two consecutive experiments are less detrimental in a laboratory environment. We demonstrate a variance of σ2 ≈4 × 10-4, lower than required for the detection of single base-pair insertion or deletion in an optofluidic chip.
RESUMO
We present an all-numerical method for post-processing of the fluorescent signal as obtained from labeled molecules by capillary electrophoresis (CE) in an optofluidic chip, on the basis of data filtering in the Fourier domain. It is shown that the method outperforms the well-known lock-in amplification during experiments in the reduction of noise by a factor of (square root)2. The method is illustrated using experimental data obtained during CE separation of molecules from a commercial DNA ladder with 17 fluorescently labeled molecules having different base-pair sizes. An improvement in signal-to-noise ratio by a factor of â¼10 is achieved, resulting in a record-low limit of detection of 210 fM.
Assuntos
DNA/análise , Filtração/métodos , Corantes Fluorescentes/química , Espectrometria de Fluorescência/métodos , Pareamento de Bases , Eletroforese Capilar/métodos , Filtração/instrumentação , Limite de Detecção , Espectrometria de Fluorescência/instrumentaçãoRESUMO
We introduce a principle of parallel optical processing to an optofluidic lab-on-a-chip. During electrophoretic separation, the ultra-low limit of detection achieved with our set-up allows us to record fluorescence from covalently end-labeled DNA molecules. Different sets of exclusively color-labeled DNA fragments-otherwise rendered indistinguishable by spatio-temporal coincidence-are traced back to their origin by modulation-frequency-encoded multi-wavelength laser excitation, fluorescence detection with a single ultrasensitive, albeit color-blind photomultiplier, and Fourier analysis decoding. As a proof of principle, fragments obtained by multiplex ligation-dependent probe amplification from independent human genomic segments, associated with genetic predispositions to breast cancer and anemia, are simultaneously analyzed.
Assuntos
DNA/análise , Dispositivos Lab-On-A-Chip , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Eletroforese/instrumentação , Eletroforese/métodos , Análise de Fourier , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sensibilidade e Especificidade , Espectrometria de FluorescênciaRESUMO
By applying integrated-waveguide laser excitation to an optofluidic chip, fluorescently labeled DNA molecules of 12 or 17 different sizes are separated by CE with high operating speed and low sample consumption of approximately 600 pL. When detecting the fluorescence signals of migrating DNA molecules with a PMT, the LOD is as low as 2.1 pM. In the diagnostically relevant size range (approximately 150-1000 base-pairs) the molecules are separated with reproducibly high sizing accuracy (> 99%) and the plug broadening follows Poissonian statistics. Variation of the power dependence of migration time on base-pair size--probably with temperature and condition of the sieving gel matrix--indicates that the capillary migration cannot be described by a simple physical law. Integrated-waveguide excitation of a 12-microm narrow microfluidic segment provides a spatio-temporal resolution that would, in principle, allow for a 20-fold better accuracy than the currently supported by state-of-the-art electrophoretic separation in microchips, thereby demonstrating the potential of this integrated optical approach to fulfill the resolution demands of future electrophoretic microchips.
Assuntos
DNA/isolamento & purificação , Eletroforese em Microchip/instrumentação , Pareamento de Bases , Eletroforese Capilar/economia , Eletroforese Capilar/instrumentação , Eletroforese em Microchip/economia , Desenho de Equipamento , Corantes Fluorescentes , Sensibilidade e EspecificidadeRESUMO
Ultrafast laser writing of waveguides in glasses is a very flexible and simple method for direct on-chip integration of photonic devices. In this work we present a monolithic optofluidic device in fused silica providing label-free and spatially-resolved sensing in a microfluidic channel. A Mach-Zehnder interferometer is inscribed with the sensing arm orthogonally crossing the microfluidic channel and the reference arm passing over it. The interferometer is integrated either with a microchannel fabricated by femtosecond laser technology or into a commercial lab-on-chip for capillary electrophoresis. The device layout, made possible by the unique three-dimensional capabilities of the technique, enables label-free sensing of samples flowing in the microchannel with spatial resolution of about 10 microm and limit of detection down to 10(-4) RIU.
RESUMO
We present a simple approach in electrophoretic DNA separation and fluorescent monitoring that allows to identify the insertion or deletion of base-pairs in DNA probe molecules from genetic samples, and to perform intrinsic calibration/referencing for highly accurate DNA analysis. The principle is based on dual-point, dual-wavelength laser-induced fluorescence excitation using one or two excitation windows at the intersection of integrated waveguides and microfluidic channels in an optofluidic chip and a single, color-blind photodetector, resulting in a limit of detection of ~200 pM for single-end-labeled DNA molecules. The approach using a single excitation window is demonstrated experimentally, while the option exploiting two excitation windows is proposed theoretically.
RESUMO
We use direct femtosecond laser writing to integrate optical waveguides into a commercial fused silica lab-on-chip (LOC). We fabricate high quality waveguides intersecting the microfluidic channels and use them to optically address with high spatial selectivity their content. Fluorescence from the photoexcited volume is efficiently collected at a 90 degrees angle by a high numerical aperture fiber, resulting in a compact and portable setup. Our approach is quite powerful because it allows the integration of photonic functionalities, by simple post-processing, into commercial LOCs, fabricated with standard techniques. By taking advantage of the unique three-dimensional capabilities of femtosecond laser writing, more complex functionalities, such as splitters or Mach-Zehnder interferometers, can be implemented.
Assuntos
Lasers , Microfluídica/instrumentação , FluorescênciaRESUMO
We use direct femtosecond laser writing to integrate optical waveguides into a commercial fused silica capillary electrophoresis chip. High-quality waveguides crossing the microfluidic channels are fabricated and used to optically address, with high spatial selectivity, their content. Fluorescence from the optically excited volume is efficiently collected at a 90 degree angle by a high numerical aperture fiber, resulting in a highly compact and portable device. To test the platform we performed electrophoresis and detection of a 23-mer oligonucleotide plug. Our approach is quite powerful because it allows the integration of photonic functionalities, by simple post-processing, into commercial LOCs fabricated with standard techniques.
Assuntos
Microfluídica/instrumentação , Óptica e Fotônica , FluorescênciaRESUMO
Using femtosecond laser writing, optical waveguides were monolithically integrated into a commercial microfluidic lab-on-a-chip device, with the waveguides intersecting a microfluidic channel. Continuous-wave laser excitation through these optical waveguides confines the excitation window to a width of 12 microm, enabling high-resolution monitoring of the passage of different types of fluorescent analytes when migrating and being separated in the microfluidic channel by microchip capillary electrophoresis. Furthermore, we demonstrate on-chip-integrated waveguide excitation and detection of a biologically relevant species, fluorescently labeled DNA molecules, during microchip capillary electrophoresis. Well-controlled plug formation as required for on-chip integrated capillary electrophoresis separation of DNA molecules, and the combination of waveguide excitation and a low limit of detection, will enable monitoring of extremely small quantities with high spatial resolution.