Letter to the Editor
A biochip reader using super critical angle fluorescence

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Abstract

We present fluorescence chip reader for rapid, multiplexed assay measurements on plastic biochips. The system is designed as a low-cost, compact and robust instrument for point of care testing. Fluorescence imaging of biochip arrays is accomplished with a custom designed piezo-motor-driven scanning stage coupled to an optical element based on supercritical angle fluorescence (SAF). The use of SAF not only provides substantial enhancement of the fluorescence collection efficiency but also confines the fluorescence detection volume strictly to the close proximity of the biochip surface, thereby discriminating against fluorescence background from the analyte solution. The high imaging resolution of the SAF scanner is demonstrated by measuring the point spread function of 200 nm fluorescence beads. The sensitivity of the system is determined by measuring an array of low fluorophore concentrations and by real-time monitoring of a model bioassay.

Introduction

Point-of-care measurements require portable and inexpensive devices, that allow physicians to conduct diagnostic tests in the surgery and that have the potential for home self-testing. These devices need to be reliable and inexpensive, while still providing sufficient sensitivity for clinically relevant analytes. Currently, a significant number of markers are available for diagnosing and analyzing the progress of diseases using immunoassays. Furthermore, key biomarkers, for example for cardiovascular disease (CVD), are increasingly used for risk monitoring for individuals and populations, in order to predict the likelihood of developing specific diseases. It is therefore desirable to be able to screen for a number of relevant analytes simultaneously on one platform for efficient risk stratification and disease diagnosis. Fluoro-immunoassays, using fluorescently labelled biomarkers, are a common method for screening and measuring analytes [1], [2]. These techniques typically involve patterned arrays of biorecognition elements which are imaged using an optical readout system. There is an increasing requirement to detect low levels of analyte in small volumes which necessitates the use of high sensitivity readout systems. Signal enhancement strategies for optical biochips include plasmonic enhancement [3], high brightness nanoparticles [4] and the use of high collection efficiency optics. In this work, the technique of supercritical angle fluorescence (SAF) is used to produce increased detection sensitivity and decreased limit of detection (LOD) compared to conventional detection systems, by substantially increasing the light collection efficiency. Moreover, SAF detection allows the collection of the fluorescence only from molecules that are in close proximity to the interface of the substrate and the sample solution and not from the bulk solution. This important aspect of SAF leads to substantial reductions in background signal. A brief description of SAF is given in the next section.

Herein, we present a compact and sensitive scanning system for multiplexed assays based on a SAF-optical element and a piezo-motor-driven scanning stage. The purpose of the design is to meet the requirements for point-of-care diagnostics. These include the ability to perform reliable and rapid measurements of multiple analytes, with sufficient sensitivity, using compact and relatively inexpensive systems. The high imaging spatial resolution of 5 μm is demonstrated here by measuring the system point spread function. The measured limit of detection was 0.14 Cy5-dye molecules per μm2 and the ability of the system to discriminate between surface and bulk detection of labelled biomolecules is also illustrated. Finally the capability of the system to perform kinetic measurements was demonstrated using a model assay system.

Section snippets

Supercritical angle fluorescence

It has been established that excited fluorescent molecules, which are in close proximity to the interface between two dielectric media, emit a large proportion of their radiation into the higher refractive index substrate. Moreover, the emitted light is highly anisotropic, with a substantial amount of fluorescence being emitted into angles above the critical angle as so-called SAF [5]. The radiation pattern can be described within the framework of electromagnetic dipole emission at a

Instrumentation of SAF-scanner

A schematic diagram of the instrument is shown in Fig. 2. The system is used to scan an array of fluorescent spots deposited on a Zeonor chip, made of an optical Cyclo Olefin coPolymer. The fluorescent spots used in this work are either Cy5 or Alexa Fluor 647 molecules or biomolecules labelled with these dyes. A laser diode (Roithner Lasertechnik, Austria) with a wavelength of 635 nm is used to excite the chip through an interference filter (FF01-625/26-25, Semrock Inc., USA) from below. Two

Chip preparation

Polymer biochips were injection moulded by Åmic AB (Uppsala, Sweden) using an optical Cyclo Olefin coPolymer (Zeonor 1060R, Zeon, Japan) resulting in disposable planar chips with a microscope slide format (75 mm × 25 mm × 1.2 mm). These chips were used in order to evaluate the optical setup as well as the scanning system. The chips were oxidized in oxygen plasma for 6 min at a working pressure of 0.26 mbar, 1000 W and with a flow of oxygen at 100 ml/min. The chips were then immersed in a solution of 3

Imaging resolution and LOD

To investigate the imaging performance of the instrument, we used fluorescent beads (200 nm, dark red 660/680-F8807, Invitrogen) as point emitters to enable determination of the instrumental point spread function. The beads were diluted in PBS to a concentration of 500 ng/ml and a droplet of 40 μl was dispensed with a pipette on an unfunctionalized Zeonor slide and this area was scanned repeatedly. Fig. 4 shows the scanned image. Each line was scanned with a step length of 1 μm. We determined the

Conclusion

We have described here a low-cost, supercritical angle fluorescence chip reader for rapid biochip analysis, which is suitable for point of care testing. The developed system exhibits a number of significant analytical performance features based primarily on exploitation of the SAF principle. In particular, the combination of the high collection efficiency and the surface selectivity of the SAF detection technique yield a generic LOD of 0.14 Cy5 fluorophores per μm2. This represents a very low

D. Kurzbuch received his PhD in 2006 from University of Zurich at the Institute for Physical Chemistry. He is currently working as a Post-Doc in the Biomedical Diagnostics Institute based at the Dublin City University in Ireland. His current interests include surface chemistry and building optical biosensors.

References (19)

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D. Kurzbuch received his PhD in 2006 from University of Zurich at the Institute for Physical Chemistry. He is currently working as a Post-Doc in the Biomedical Diagnostics Institute based at the Dublin City University in Ireland. His current interests include surface chemistry and building optical biosensors.

J. Bakker was born in 1976 in Kootwijkerbroek, the Netherlands. He received MSc in applied physics in 2001 at Twente University, Enschede, the Netherlands, with a specialization in Rheology. In 2001, he started a research position at Linköping University, Sweden, in the Laboratory of Applied Optics. There he has worked on improvement of porous silicon-based gas sensors by polymer modification, determination of the refractive index of printed and unprinted papers using spectroscopic ellipsometry and fluorimetry and ellipsometry using computer screen photo-assisted techniques. He obtained his PhD in 2006 and is currently working as a Post-Doc in the Biomedical Diagnostics Institute based at Dublin City University in Dublin Ireland (employed by Åmic AB, Uppsala, Sweden), where he is working on optically enhanced fluorescence based point-of-care instrumentation.

J. Melin received a diploma in biological research from Cardiff University (UK) in 2000, a MSc in Engineering Biology from Linköping University (Sweden) in 2002 and a PhD in Engineering Science from Uppsala University (Sweden) in 2006. He is currently employed by Åmic AB (Uppsala, Sweden) as an industry Post-Doc and is stationed at the Biomedical Diagnostics institute in Dublin (Ireland). His research interests involve polymer micro fabrication techniques, microfluidics, single molecule detection methods and chip-based immunoassays.

C. Jönsson received her PhD in Organic Chemistry from The Royal Institute of Technology, Stockholm, and she has a post doctoral position at Åmic AB, Sweden/Dublin City University. Her current interests include medicinal, organic and surface chemistry used in diagnostics and for biosensors.

T. Ruckstuhl received a diploma in physics from the University of Heidelberg (Germany) in 1997 and a PhD in biophysics from the University of Regensburg (Germany) in 1999. He worked on SAF based biosensing and microscopy at the University of Zurich in Switzerland (1999–2004) and at the Dublin City University in Ireland (2004–2007). Since 2007 he is employed as a senior research fellow at the University of Zurich. His research interests involve near- and far-field microscopy, biosensors, fluorescence methods, optical engineering, microfluidics, immunoassays, design of biochips and optical well plates.

Brian MacCraith is professor at Dublin City University (DCU) and is an expert in the field of optical chemical sensors and biosensors. In 1999 he was appointed director of the National Centre of Sensor Research (NCSR) at DCU. His work has focused mainly on the exploitation of luminescence techniques in combination with advanced photonic materials and innovative measurements platforms with particular application to array biochips. In 2005 he left NCSR to become institute director at the Biomedical Diagnostics Institute at DCU.

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