Oliver Hofmann

Monolithically integrated dye-doped PDMS long-pass filters for disposable on-chip fluorescence detection

We report the fabrication of high quality monolithically integrated optical long-pass filters, for use in disposable diagnostic microchips. The filters were prepared by incorporating dye molecules directly into the microfluidic chip substrate, thereby providing a fully integrated solution that removes the usual need for discrete optical filters. In brief, lysochrome dyes were added to a poly(dimethylsiloxane) (PDMS) monomer prior to moulding of the microchip from a structured SU-8 master. Optimum results were obtained using 1 mm layers of PDMS doped with 1200 microg mL(-1) Sudan II, which resulted in less than 0.01% transmittance below 500 nm (OD 4), >80% above 570 nm, and negligible autofluorescence. These spectral characteristics compare favourably with commercially available Schott-glass long-pass filters, indicating that high quality optical filters can be straightforwardly integrated into the form of PDMS microfluidic chips. The filters were found to be robust in use, showing only slight degradation after extended illumination and negligible dye leaching after prolonged exposure to aqueous solutions. The provision of low cost high quality integrated filters represents a key step towards the development of high-sensitivity disposable microfluidic devices for point-of-care diagnostics.

Integrated thin-film polymer/fullerene photodetectors for on-chip microfluidic chemiluminescence detection

We report the use of solution-processed thin-film organic photodiodes for microscale chemiluminescence. The active layer of the photodiodes comprised a 1 : 1 blend by weight of the conjugated polymer poly(3-hexylthiophene) [P3HT] and [6,6]-phenyl-C(61)-butyric acid-methylester [PCBM]--a soluble derivative of C(60). The devices had an active area of 1 mm x 1 mm, and a broad-band response from 350 to 700 nm, with an external quantum efficiency of more than 50% between 450 and 550 nm. The photodiodes have a simple layered structure that permits facile integration with planar chip-based systems. To evaluate the suitability of the organic devices as integrated detectors for microscale chemiluminescence, a peroxyoxalate based chemiluminescence reaction (PO-CL) was monitored within a poly(dimethyl-siloxane) (PDMS) microfluidic device. Quantitation of hydrogen peroxide indicated excellent linearity and yielded a detection limit of 10 microM, comparable with previously reported results using micromachined silicon microfluidic chips with integrated silicon photodiodes. The combination of organic photodiodes with PDMS microfluidic chips offers a means of creating compact, sensitive and potentially low-cost microscale CL devices with wide-ranging applications in chemical and biological analysis and clinical diagnostics.

Towards microalbuminuria determination on a disposable diagnostic microchip with integrated fluorescence detection based on thin-film organic light emitting diodes.

As a first step towards a fully disposable stand-alone diagnostic microchip for determination of urinary human serum albumin (HSA), we report the use of a thin-film organic light emitting diode (OLED) as an excitation source for microscale fluorescence detection. The OLED has a peak emission wavelength of 540 nm, is simple to fabricate on flexible or rigid substrates, and operates at drive voltages below 10 V. In a fluorescence assay, HSA is reacted with Albumin Blue 580, generating a strong emission at 620 nm when excited with the OLED. Filter-less discrimination between excitation light and generated fluorescence is achieved through an orthogonal detection geometry. When the assay is performed in 800 microm deep and 800 microm wide microchannels on a poly(dimethylsiloxane)(PDMS) microchip at flow rates of 20 microL min(-1), HSA concentrations down to 10 mg L(-1) can be detected with a linear range from 10 to 100 mg L(-1). This sensitivity is sufficient for the determination of microalbuminuria (MAU), an increased urinary albumin excretion indicative of renal disease (clinical cut-off levels: 15-40 mg L(-1)).

Modular approach to fabrication of three-dimensional microchannel systems in PDMS—application to sheath flow microchips

A modular approach to fabrication of three-dimensional microchannel systems in polydimethylsiloxane (PDMS) is presented. It is based on building blocks with microstructuring on up to three faces. The assembled 3D-microchip consists of three building blocks in two layers. For assembly of the bottom layer two building blocks are joined horizontally, whereby the side structuring of the first is sealed against the flat side surface of the other. This results in the formation of a vertical interconnection opening between the building blocks to supplement the microstructuring on the lower faces. The 3D microchannel system is completed by placing a third building block, with microstructuring only on its lower face, on top of the assembled layer. While plasma assisted bonding is used between the two building blocks of the bottom layer, inherent adhesion is sufficient between the layers and for attaching the assembled 3D-microchip to a substrate. This modular approach was applied to the fabrication of a 3D-sheath flow microchip. It comprises a 20 microm deep microchannel system with sample inlet, open sensing area and outlet in the bottom layer and sheath flow inlet in the top layer. 100 microM fluorescein at 6 microL min(-1) was used as sample flow and water at increasing flow rates as sheath flow. With ratios of sheath to sample flow up to 20:1 sample layers down to 1 microm thickness could be generated. Sample layer thickness was determined via volume detection on an epi-fluorescence microscope followed by image analysis.

Thin-film polymer light emitting diodes as integrated excitation sources for microscale capillary electrophoresis

We report the use of a thin-film polymer light emitting diode as an integrated excitation source for microfabricated capillary electrophoresis. The polyfluorene-based diode has a peak emission wavelength of 488 nm, an active area of 40 microm x 1000 microm and a thickness of similar 2 mm. The simple layer-by-layer deposition procedures used to fabricate the polymer component allow facile integration with planar chip-based systems. To demonstrate the efficacy of the approach, the polyfluorene diode is used as an excitation source for the detection of fluorescent dyes separated on-chip by electrophoresis. Using a conventional confocal detection system the integrated pLED is successfully used to detect fluorescein and 5-carboxyfluorescein at concentrations as low as 10(-6) M with a mass detection limit of 50 femtomoles. The drive voltages required to generate sufficient emission from the polymer diode device are as low as 3.7 V.

Patterning of organic devices by interlayer lithography

We report a new lithographic procedure that enables the patterning of as-received semiconducting polymers and small molecules at the near micron level without causing discernible degradation of the patterned material. The method involves a minimum of processing steps, requires no modification of the active layer, and is compatible with both rigid and flexible substrates. The technique makes use of an intermediate resist layer between the substrate and the active layer, i.e.underneath the active layer, and involves the simultaneous patterning of the resist and active layers in a single expose/develop step. The technique has been successfully applied to the fabrication of flexible ITO-free light-emitting diodes and photodiodes, yielding peak quantum efficiencies of 8.8 cd A−1 and 57% respectively comparable to similar devices fabricated on ITO-coated glass. It is also readily extendible to the patterning on a single substrate of multiple devices incorporating different component materials, e.g. the red, green and blue pixels of a colour display.

Time-resolved fluorescence imaging of solvent interactions in microfluidic devices

We present the application of wide-field time-resolved fluorescence imaging methods for the study of solvent interactions and mixing in microfluidic devices. Time-resolved imaging of fluorescence polarization anisotropy allows us to image the local viscosity of fluorescence in three dimensions in order to directly monitor solvent mixing within a microfluidic channel. This provides a viscosity image acquisition time of the order of minutes, and has been applied to a steady-state laminar flow configuration. To image dynamic fluid mixing in real-time, we demonstrate high-speed fluorescence lifetime imaging at 12.3 Hz applied to DASPI, which directly exhibits a solvent viscosity-dependant fluorescence lifetime. These two methods facilitate a high degree of quantification of microfluidic flow in 3-D and/or at high speed, providing a tool for studying fluid dynamics and for developing enhanced microfluidic assays.

Laser induced disruption of bacterial spores on a microchip

We report on the development of a laser based spore disruption method. Bacillus globigii spores were mixed with a laser light absorbing matrix and co-crystallized into 200-microm-wide and 20-microm-deep nanovials formed in a polydimethylsiloxane (PDMS) target plate. Surface tension effects were exploited to effect up to 125-fold spore enrichment. When the target zones were illuminated at atmospheric pressure with pulsed UV-laser light at fluences below 20 mJ cm(-2) a change in spore morphology was observed within seconds. Post illumination PCR analysis suggests the release of endogenous DNA indicative of spore disruption. For laser fluences above 20 mJ cm(-2), desorption of spores and fragments was also observed even without a matrix being employed. Desorbed material was collected in a PDMS flowcell attached to the target plate during laser illumination. This opens up a route towards the direct extraction of released DNA in an integrated spore disruption-PCR amplification microchip device.

Organic light emitting diodes and photodetectors: Toward applications in lab-on-a-chip portable devices

We report that polymer light emitting diodes (pLEDs) and polymer photodetectors can be integrated on disposable polydimethylsiloxane [PDMS] microfluidic flowcells to form hybrid microchips for bioluminescence applications. PLEDs were successfully employed as excitation light sources for microchip based fluorescence detection of microalbuminuria (MAU), an increased urinary albumin excretion indicative of renal disease. To circumvent the use of optical filters, fluorescence was detected perpendicular to the biolabel flow direction using a CCD spectrophotometer. Prior to investigating the suitability of polymer photodiodes as integrated detectors for fluorescence detection, their sensitivity was tested with on-chip chemiluminescence. The polymer photodetector was integrated with a PDMS microfluidic flowcell to monitor peroxyoxalate based chemiluminescence (CL) reactions on the chip. This work demonstrates that our polymer photodetectors exhibit sensitivities comparable to inorganic photodiodes. Here we prove the concept that thin film solution-processed polymer light sources and photodetectors can be integrated with PDMS microfluidic channel structures to form a hybrid microchip enabling the development of disposable low-cost diagnostic devices for point-of-care analysis.

Chapter 4 Multidimensional fluorescence imaging

Publisher Summary This chapter describes the applications of multidimensional fluorescence imaging (MDFI) instrumentation. The chapter discusses fluorescence lifetime imaging (FLIM), with an emphasis on rapid wide-field time-gated imaging, including application to molecular biology, FLIM of tissue autofluorescence, and highspeed optically sectioned FLIM for live cell imaging. Optical sectioning is important to enhance image contrast and to minimize unwanted mixing of signals from axially separate fluorophores. The chapter discusses the extension to spectral FLIM, including (emission resolved) hyperspectral FLIM, implemented using line-scanning microscopy, and excitation-resolved imaging and FLIM utilizing supercontinuum generation to provide excitation throughout the visible spectrum. The combination of polarization-resolved and time resolved fluorescence imaging is described, mapping both lifetime and rotational correlation time as illustrated by an application to microfluidic devices. This chapter demonstrates that FLIM and MDFI can add significant value to microscopy, endoscopy, and assay technology. By resolving the fluorescence signal with respect to multiple dimensions including excitation and emission wavelength, lifetime, and polarization, it may be possible to enhance the fluorescence read-out for an experiment or assay, for example, in terms of improving the separation of multiple fluorophores or imaging variations in the local molecular environment.