Laminar Flow Microarray Patterning by Perpendicular Electrokinetic Focusing

This paper describes a method to pattern microarrays in a closed microfluidic device. Two perpendicular laminar flow streams can operate in terms to sequentially coat the surface of a flow-chamber with parallel lanes in two directions. Two perpendicular sample streams can be controlled in position and width by applying electrokinetic focusing, for which each of the two streams is sandwiched between two parallel laminar flow streams containing just a buffer solution. Electroosmotic flow allows a simple chip design without any moving parts being involved. With this device configuration it is possible to define an array of up to 169 spots on a surface area of 1 mm2

Signal amplification on planar and gel-type sensor surfaces in surface plasmon resonance-based detection of prostate-specific antigen

This article describes surface plasmon resonance (SPR)-based detection of prostate-specific antigen (PSA), comparing amplification with colloidal gold (10 nm diameter) and latex microspheres (120 nm diameter) on planar- and gel-type sensor surfaces. As matrix, 3% BSA in PBS was used. Experimental data were compared with model calculations that predict the SPR signal that results from covering of the different sensor surfaces with each of the particles used. Amplification with latex particles gave a higher signal than did that with colloidal gold. However, the limit of detection (LOD) attained by latex amplification was not as good as that obtained after gold amplification, and this was unexpected. LOD and sensitivity of the amplified PSA assays when performed with the planar-type sensor disc were equally good or better compared with those when performed with the gel-type sensor disc. Indirect evidence indicates a restricted accessibility of the gel layer on the gel-type sensor toward the colloidal gold. Application of colloidal gold led to a sensitivity increase of approximately three orders of magnitude compared with nonamplified detection. The corresponding LOD was approximately 0.15 ng PSA/ml, which is sufficient for measuring enhanced, clinically relevant PSA levels (>4 ng/ml)

Multi-wavelength fluorescence sensing with integrated waveguides in an optofluidic chip

Femtosecond-laser-written integrated waveguides enable multi-wavelength fluorescence sensing of flowing biomolecules in an optofluidic chip. Fluorescence from differently labeled biomolecules with distinct absorption wavelengths, encoded by uniquely modulating each excitation beam, is detected by a color-blind photodetector, and the origin of each signal is unraveled by Fourier analysis

Multi-color fluorescent DNA analysis in an integrated optofluidic lab-on-a-chip

Sorting and sizing of DNA molecules within the human genome project has enabled the genetic mapping of various illnesses. By employing tiny lab-on-a-chip devices for such DNA analysis, integrated DNA sequencing and genetic diagnostics have become feasible. However, such diagnostic chips typically lack integrated sensing capability. We address this issue by combining microfluidic capillary electrophoresis with laser-induced fluorescence detection resulting in optofluidic integration towards an on-chip bio-analysis tool [1,2]. We achieve a spatial separation resolution of 12 μm, which can enable a 20-fold enhancement in electropherogram peak resolution, leading to plate numbers exceeding one million. We demonstrate a high sizing/calibration accuracy of 99% [3], and ultrasensitive fluorescence detection (limit of detection = 65 femtomolar, corresponding to merely 2-3 molecules in the excitation/detection volume) of diagnostically relevant double-stranded DNA molecules by integrated-waveguide laser excitation. Subsequently, we introduce a principle of parallel optical processing to this optofluidic lab-on-a-chip. Different sets of exclusively color-labeled DNA fragments – otherwise rendered indistinguishable by their spatio-temporal coincidence – are traced back to their origin by modulation-frequency-encoded multi-wavelength laser excitation, fluorescence detection with a color-blind photomultiplier, and Fourier-analysis decoding. As a proof of principle, fragments from independent human genomic segments, associated with genetic predispositions to breast cancer and anemia, are extracted by multiplex ligation-dependent probe amplification, and simultaneously analyzed. Such multiple yet unambiguous optical identification of biomolecules opens new horizons for “enlightened” lab-on-a-chip devices

Fluorescence monitoring of capilarry electrophoresis separation in a lab-on-a-chip with monolithically integrated waveguides

Femtosecond-laser-written optical waveguides were monolithically integrated into a commercial lab-on-a-chip to intersect a microfluidic channel. Laser excitation through these waveguides confines the excitation window to a width of 12 μm, enabling high-spatial-resolution monitoring of different fluorescent analytes, during their migration/separation in the microfluidic channel by capillary electrophoresis. Wavelength-selective monitoring of the on-chip separation of fluorescent dyes is implemented as a proof-of-principle. We envision well-controlled microfluidic plug formation, waveguide excitation, and a low limit of detection to enable monitoring of extremely small quantities with high spatial resolution

Multi-point, multi-wavelength fluorescence monitoring of DNA separation in a lab-on-a-chip with monolithically integrated femtosecond-laser-written waveguides

Electrophoretic separation of fluorescently labeled DNA molecules in on-chip microfluidic channels was monitored by integrated waveguide arrays, with simultaneous spatial and wavelength resolution. This is an important step toward point-of-care diagnostics with multiplexed DNA assays

Fluorescence monitoring of capillary electrophoresis separation of biomolecules with monolithically integrated optical waveguides

Monolithic integration of optical waveguides in a commercial lab-on-a-chip by femtosecond-laser material processing enables arbitrary 3D geometries of optical sensing structures in combination with fluidic microchannels. Integrated fluorescence monitoring of molecular separation, as applicable in point-of-care diagnostic bioassays is demonstrated

Monitoring of DNA molecules in a lab on a chip with femtosecond laser written waveguides

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 um, 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. We demonstrate on-chip-integrated waveguide excitation and detection of a biologically relevant species, fluorescently labeled Deoxyribonucleic acid (DNA) molecules, as well as separation of different dyes, Rhodamine-6G and Rhodamine-B 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 detection limit will enable monitoring of extremely small quantities with high spatial resolution

Integrated fluorescence sensing in a lab-on-a-chip for DNA analysis

We report on monolithic optical sensor integration in a lab-on-a-chip toward onchip diagnosis of all kinds of genetic diseases, e.g. breast cancer. Such an analysis of genetic diseases is based on the capillary electrophoresis separation of DNA fragments amplified from diagnostically relevant regions of the concerned gene.\ud This paper presents a proof of principle, demonstrating real time integrated fluorescence monitoring during the capillary electrophoresis separation of fluorescent dyes as well as fluorescently labeled DNA molecules in an on-chip microfluidic channel. To this end, sensing waveguides were integrated monolithically by means of femtosecond laser irradiation, in a commercial fused silica lab-on-a-chip, to perpendicularly intersect the microfluidic separation channel. Laser excitation through these waveguides induces fluorescence in the flowing microfluidic plugs. Depending on the number of species present, and the difference between their mobility, a corresponding number of electropherogram peaks are detected. \ud Detection has been performed by a CCD camera in order to visualize the on-chip events and to provide access to spatial information, as well as by a photomultiplier tube in order to detect low values of fluorescence signal. The present limit of detection is estimated to be approximately 6 nM. The presented setup achieves high spatial resolution due to the small cross section of the waveguides, ~12 μm. This is an improvement over the conventional approach to place a pinhole in the path of the fluorescence output signal generated by a broadband background illumination, e.g. with an Hg or a Xe lamp. Future work will focus on extension of this principle to real world diagnostic samples for development of a fast and compact point of care optical biosensing device.\ud During the conference we will present the latest results in wavelengthmultiplexed fluorescence monitoring of DNA separation at low limits of detection