Biomedical Microdevices

, Volume 13, Issue 4, pp 759–767

Novel disposable biochip platform employing supercritical angle fluorescence for enhanced fluorescence collection

Authors

  • Duncan Hill
    • Biomedical Diagnostics InstituteDublin City University
  • Barry McDonnell
    • Biomedical Diagnostics InstituteDublin City University
  • Stephen Hearty
    • Biomedical Diagnostics InstituteDublin City University
  • Lourdes Basabe-Desmonts
    • Biomedical Diagnostics InstituteDublin City University
  • Robert Blue
    • Biomedical Diagnostics InstituteDublin City University
  • Michal Trnavsky
    • Biomedical Diagnostics InstituteDublin City University
    • Biomedical Diagnostics InstituteDublin City University
  • Richard O’Kennedy
    • Biomedical Diagnostics InstituteDublin City University
  • Brian D. MacCraith
    • Biomedical Diagnostics InstituteDublin City University
Article

DOI: 10.1007/s10544-011-9546-2

Cite this article as:
Hill, D., McDonnell, B., Hearty, S. et al. Biomed Microdevices (2011) 13: 759. doi:10.1007/s10544-011-9546-2

Abstract

This paper presents an overview of development of a novel disposable plastic biochip for multiplexed clinical diagnostic applications. The disposable biochip is manufactured using a low-cost, rapid turn- around injection moulding process and consists of nine parabolic elements on a planar substrate. The optical elements are based on supercritical angle fluorescence (SAF) which provides substantial enhancement of the fluorescence collection efficiency but also confines the fluorescence detection volume strictly to the immediate proximity of the biochip surface, thereby having the potential to discriminate against background fluorescence from the analyte solution. An optical reader is also described that enables interrogation and fluorescence collection from the nine optical elements on the chip. The sensitivity of the system was determined with a biotin-avidin assay while its clinical utility was demonstrated in an assay for C-reactive protein (CRP), an inflammation marker.

Keywords

Lab on a chipSupercritical angle fluorescenceImmunoassayCardiac marker

1 Introduction

There is a strong demand for the development of disposable biochips for point-of-care (POC) diagnostic assay platforms, for use in a broad range of settings. Such miniaturised and inexpensive biochips are designed to migrate the analysis from the hospital laboratory to the POC setting, where the complicated sequences of clinical tests can be performed quickly by relatively un-skilled personnel (Ligler 2009). Common transduction technologies employed in POC immunoassay platforms have traditionally been optically-, or electrochemically-mediated (Soper et al. 2006). Fluorescence-based systems are the most widely used due primarily to their superior sensitivity compared with alternative approaches. In such systems, fluorescent labels are employed to transduce the biomolecular binding event (Schult et al. 1999). In addition, fluorescence-based methodologies are ideally suited to parallel detection, often referred to as array or imaging detection (Cho and Bright 2002). The use of arrays enables the detection of multiple analytes in a single test. Such tests generally consist of multiple bio-recognition elements patterned onto a surface, which is typically planar or has planar elements to facilitate both patterning and detection. There is an increasing requirement to detect low levels of analyte in small volumes and this necessitates the use of high sensitivity detection techniques. Signal enhancement strategies that may be employed for optical biochips include plasmonic enhancement (Nooney et al. 2010; Stranik et al. 2007), high-brightness nanoparticles (Nooney et al., 2009) and the use of high collection efficiency optics.

Supercritical Angle Fluorescence (SAF) refers to the anisotropic emission of dipoles near a dielectric interface characterised by a preferential emission into angles above the critical angle in a substrate with higher refractive index than the superstrate (Lukosz and Kunz 1977; Enderlein et al. 1999; Ruckstuhl et al. 1999; Polerecky et al. 2002; Polerecky et al. 2000; Blue et al. 2005). Exploitation of this near-field optical effect enables surface-confined fluorescent species, bound or in close proximity (~ λ) to a substrate, to be collected efficiently by customised optics onto a detector. In particular, it has been demonstrated that paraboloid elements enable detection of fluorescence down to ultra-low concentrations (Verdes et al. 2007; Ruckstuhl et al. 2000). Typically a paraboloid element is built into an optical system, which, when optically-coupled to a planar substrate, enables efficient collection of surface-localised fluorescence. For example, efficient multiplexed assay measurements on plastic biochips via a scanning paraboloid format have been reported (Kurzbuch et al. 2009). The combination of the total internal reflection (TIR) excitation technique with a parabolic lens for SAF collection has been shown to give an attoliter detection volume and single molecule sensitivity (Ruckstuhl and Seeger 2004). It has also been shown that with only SAF collection using a parabolic element (and an aspheric lens below or an objective lens above focusing excitation light) one can achieve diffraction-limited high aperture collection of fluorescence down to the level of even a single molecule (Verdes et al. 2007; Ruckstuhl and Verdes 2004).

The work presented here is the first report of a novel embodiment for low-cost disposable plastic array biochips whereby the paraboloid elements are integrated into the biochips. In the particular example presented, the biochip comprises an array of nine paraboloid elements. The nine elements are designed for highly localised excitation (via TIRF) and highly efficient collection of light (SAF) from the planar interface. A simple optical reader employing efficient excitation and detection is also presented. The relative sensitivity of the system was determined using a dye-labelled biotin-neutravidin direct binding assay. As a proof of principle, the clinical utility of the biochip was demonstrated with an assay for the cardiac marker C-reactive protein (CRP), an indicator for inflammation (McDonnell et al. 2009).

1.1 Super-critical angle fluorescence (SAF)

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 (Hellen and Axelrod 1987). Fundamental modelling of an oscillating dipole, close to an interface of higher refractive index material, reveals that the emitted light radiates in a highly anisotropic manner (Lukosz and Kunz 1977; Enderlein et al. 1999; Ruckstuhl et al. 1999; Polerecky et al. 2002; Polerecky et al. 2000; Blue et al. 2005). The anisotropic emission results in a significant part of the fluorescence being emitted directly into the substrate of higher refractive index at angles above the critical angle for total internal reflection (Fig. 1(a)). This is called SAF emission. By virtue of its emission angles, the SAF light is trapped in a substrate with parallel planar surfaces and is therefore not available for detection above or below the chip (Fig. 1(a)). There are a number of techniques for overcoming this problem and for accessing the trapped SAF radiation. An integrated paraboloid element provides a highly efficient optical geometry to collect this surface–localised emission (Fig. 1(b)). The paraboloid element is perfectly suited to convert the high angle emission into collimated rays along the optical axis. Only fluorescence emitted close to the interface emits at angles above the critical angle. Consequently, exclusive collection of SAF strictly confines the detection volume to the surface. By placing an annular aperture (Fig. 1(c)) below the paraboloid element, one can prevent detection of fluorescence that has been emitted into the substrate at angles lower than θc. This is termed non-SAF radiation. The surface confinement of SAF is comparable to the widely used total internal reflection fluorescence (TIRF) technique. An additional attractive feature of the configuration proposed here is that the paraboloid element-annular mask combination can both excite the fluorescence above the critical angle and thereby accomplish TIRF excitation, whilst simultaneously capturing the SAF emission (Fig. 2 (a) and (b)).
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Fig. 1

(a) SAF fluorescence trapped in a planar substrate due to total internal reflection (b) Polar diagram of the angular distribution of emission of surface-bound fluorophores with isotropic dipole orientation. A paraboloid reflector converts large-angle fluorescence into collimated light. (c) Schematic of captured surface angles. The annular mask enables selection of all, or a defined range of, SAF angles

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Fig. 2

Combined paraboloid and annular mask platform that (a) redirects collimated incoming light onto the bio-detection surface above critical angle in order to produce surface-confined TIRF excitation and (b) the same platform exclusively collects SAF fluorescence above the critical angle

2 Materials and methods

2.1 Paraboloid biochip

A nine element paraboloid biochip (Fig. 3(a)) was injection moulded (Nalux Co. Ltd, Japan) using a cyclo olefin copolymer (Zeonex, Zeon Corporation, Japan). Zeonex was chosen due to its excellent optical properties and low auto-fluorescence in order to minimise sources of background noise. The biochip comprised a 3 × 3 array of integrated paraboloids, each of which was 0.8 mm in height. The diameter of each paraboloid was 1.8 mm at the top and 3 mm at the base (Fig. 3(b)). Each paraboloid has two parallel plane faces. The geometrical focus of the parabolic surface lies in the center of the smaller planar face, the top face, and represents the point of collection of surface generated fluorescence. The parabolic surface of the element acts as a loss-free mirror via total internal reflection. The geometry of the paraboloid can be described by z = αx2, 0 > zfrontzzrear, where z denotes the optical axis of the system, zfront the position of the top face, zrear the position of the bottom face and α is a given constant which in this case is 0.55. The z-position of the front face of the paraboloid is chosen in such a way that the focal point of the collector lies exactly at the top surface of each paraboloid.
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Fig. 3

(a) Parabolic chip array; (b) Chip and parabolic element dimensions in millimetres; (c) Annular aperture mask for blocking non-TIRF excitation and non-SAF emission

The chips were coated with a proprietary surface chemistry comprising an aldehyde-activated dextran surface coating for antibody immobilisation (Åmic AB, Uppsala, Sweden). The resultant aldehyde surface enabled immobilization of amino-functionalized molecules, such as proteins, onto the surface. Reaction vessels used in surface discrimination experiments were made from Poly(methylmethacrylate) (PMMA). Nine wells, 2 mm in diameter, were micro-milled into a 2 mm thick piece of PMMA. Each well was aligned to a matching paraboloid element. The PMMA was attached to the chip via 50 μm thick pressure-sensitive-adhesive (ARcare®8890, Adhesives Research, Ireland).

2.2 Biochip reader

The optical set-up is shown schematically in Fig. 4. A 5 mW laser diode (HL633G, Thorlabs, UK) with a 635 nm emission wavelength and nearly circular output beam was mounted within a tube behind a collimating achromatic lens (focal length 50 mm) and excitation filter (FF01-624/40, Semrock). The collimated beam was reflected by a dichroic mirror (z635rdc, Semrock) and redirection mirror through the annular aperture mask and onto the parabolic biochip platform via an array of annular apertures. Emitted fluorescence was reflected by the sides of the paraboloid element and travelled back through the annular mask, the dichroic mirror and through an emission filter (HQ700/75 m, Semrock). It was then focused by a pair of achromatic lenses (focal lengths 75 mm and 30 mm, with diameter of 25 mm) onto a small USB-controlled CCD camera (SXVM7, Starlight Xpress, UK). The annular mask (Fig. 3(c)) was placed below the chip to eliminate both excitation light passing vertically through the upper surface but also to block non-SAF emission. The system was linked to a laptop computer and custom software was developed in Visual Basic for image capture and analysis.
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Fig. 4

Schematic representation of the optical reader for the array biochip

2.3 Sensitivity assay

Commercial neutravidin (Pierce, #31000) was diluted in 50 mM sodium phosphate buffer, pH 7.5 containing 1% (w/v) milk powder (spotting solution) to a final concentration of 200 μg/mL and deposited onto each paraboloid element using a sciFLEXARRAYER™ piezo-dispensing system (Scienion, Germany). Following 1 h incubation in 70% humidity at room temperature, the spotted chips were blocked in a solution of phosphate buffered saline (PBS) containing 5% (w/v) milk powder overnight at 4°C. Excess blocking solution from the incubation was removed by washing with PBS. Atto 647 Biotin (Sigma, #30700), a fluorescently-labelled biotin conjugate, was then diluted in PBS containing 0.05% (v/v) tween20 and 0.5% (w/v) milk powder to concentrations ranging from 10 μM to 10 M. A 350 μL aliquot of each of the dilutions was applied to the chips and incubated for 1 h at room temperature. Next, any unbound biotin was removed by washing in PBS containing 0.05% (v/v) Tween20 (PBST) three times and then in PBS three times, followed by a wash with deionised water to remove any excess salt. The chips were then read in the custom-built biochip reader.

2.4 Anti-CRP polyclonal antibody production, purification and labelling

A New Zealand White rabbit was subjected to a comprehensive immunisation regimen with CRP as the antigen (AMS Biotechnogy, A97201H). Following several boosts, a significant response to the CRP antigen was confirmed via titration of the rabbit serum. The polyclonal antibody (pAb) was then purified from the rabbit serum using protein A affinity chromatography. A sample of the purified CRP-specific polyclonal antibody was fluorescently labelled using a commercially-available kit (DyLight 647 monoclonal antibody labelling kit, Pierce #53015).

2.5 CRP assay

Purified rabbit anti-CRP pAb (capture antibody) was diluted in spotting buffer to a final concentration of 200 μg/mL. The pAb solution was then accurately deposited as described above. Following a 1 h incubation in 70% humidity at room temperature, the spotted chips were blocked and washed as described above for the biotin capture assay. Next, varying concentrations of CRP, diluted in CRP-depleted serum (HyTest, 8CFS), were pipetted onto each of the washed chip surfaces (330 μL per chip). Following a 1 h incubation at room temperature, another wash step was performed comprising PBST (x3) and PBS (x3) to remove excess unbound CRP antigen. A 1/400 dilution of fluorescently labelled anti-CRP pAb in 1% (w/v) milk-PBST was then pipetted onto the chip surface and left to incubate for 1 h at room temperature. A final wash step was then performed as described earlier, followed by a wash with deionised water, to remove any excess salt. Finally, the washed chips read using the custom-built biochip reader.

3 Results and discussion

3.1 Chip and reader characterisation

A series of 10 aldehyde-dextran-coated biochips were spotted with Cy5-labelled human-IgG as described above. A 1 μg/mL concentration of Cy5-labelled IgG antibody was spotted into the centre of the paraboloids on all ten chips and then washed and blocked leaving only surface-immobilised Cy5-labelled antibodies on the surface. These chips were scanned in a conventional fluorescence scanner, GMS 418 ArrayScanner (Genetic MicroSystems) and the fluorescence spots quantified to ensure the uniformity of immobilised antibody on the top surface of each parabola. The standard deviation of fluorescence measured from each parabola across all ten chips was found to be within 2%, showing that uniform deposition of protein onto the tops of the paraboloids is possible.

The same chips were then placed in the optical reader and analysed over an integration time of 0.2 s. To interrogate the chip, a collimated excitation beam (Fig. 2(a)) is incident on chip from below. The annular ring allows light to be incident only on the paraboloid walls of each element on the chip and not directly onto the paraboloid centre. This light is redirected by the walls (by total internal reflection) onto the centre point of each paraboloid element at angles that generate evanescent wave excitation of fluorophores on the element surface. Light from surface-bound fluorophores is emitted preferentially into supercritical angles (Fig. 2(b)), and is redirected again by the paraboloid walls through the annular apertures into a ‘loss-free’ collimated beam towards a detector. The collimated light from the chip was imaged onto the CCD camera and the fluorescence rings (Fig. 5(a)) coming from each paraboloid analysed by appropriate software. The magnitude of the captured SAF signal is directly proportional to the number of fluorophores on the surface.
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Fig. 5

(a) Image showing collected SAF fluorescence from nine paraboloid elements. Rings around the edges are distorted due to spherical aberration and the Gaussian nature of the excitation beam; (b) Image output of fluorescence, showing SAF and non-SAF components. Rings 3, 5, 6, 8 & 9 show just the SAF rings. For rings 1, 2, 4 & 7 the annular mask has been removed. The non-SAF component appears in the centre of these particular rings

A reference chip was made by spotting a 1 μg/mL of Cy5-labelled human IgG onto the tops of all parabolas. The SAF images captured from this reference chip show that the outer rings are distorted (Fig. 5(a)). This is due to spherical aberration caused by the achromatic doublet used for focusing the ring array down onto the CCD sensor. Consistent with POC diagnostics, low cost optical elements have been used in this system. This effect can be eliminated by using higher cost, larger numerical aperture lenses. The integrated fluorescent intensities of the outer rings are also lower than that of the centre ring. This is due to the Gaussian beam profile of the laser beam incident on the chip resulting in non-uniform excitation of the fluorescent dye on the surface of the differing paraboloids. In order to correct for this, all the grey-scale images were processed and analysed using Cell^f fluorescence imaging software (Olympus, UK). Each ring was isolated and the average fluorescence intensity was measured. Normalization coefficients for each paraboloid were determined from the differences in fluorescence, measured from each paraboloid element, with respect to the central ring, which was the brightest ring. These coefficients were applied to all subsequent measurements.

The background signal in each image has two principal contributory elements; a residual capacitance in the CCD and thermal noise in the CCD; the former is invariant to around 0.1% and the latter dependent upon integration time. To account for this, the signal from a blank aldehyde-dextran-coated chip was measured before each experimental run in order to provide a systematic background measurement. This average background signal was then subtracted from all measured data before further processing.

To demonstrate the enhanced collection efficiency of the paraboloid elements, signals from the central paraboloid of chips used in the biotin-avidin assay were measured in the optical reader with and without the annular masks in place. This allowed both direct illumination of selected paraboloid surfaces in addition to TIRF excitation but also enabled fluorescence emitted into the lower angles (non-SAF) pass through to the detector via the pair of achromatic lenses. Ray-tracing analysis (TracePro, Lambda Research Corp., USA) of the system showed that the non-SAF light was collected in a small spot at the centre of the SAF image rings. While all of the light from the SAF component was collected due to the paraboloid element, the non-SAF component remained divergent and, thus, only a portion of the non-SAF light was collected, equating to about 10% of the total non-SAF component. Figure 5(b) shows an example of the resultant images collected. Visually it is obvious that the SAF signal from the rings are much greater than the non-SAF signal coming from the central spot. The SAF and non-SAF components could be isolated and analysed as described above. The signal was integrated for each ring and spot, the normalisation coefficient applied and the background signal was subtracted from the final value. The analysis shows that the paraboloid elements were able to collect between 80 and 100 times more SAF fluorescence than the non-SAF fluorescence. This is in agreement with theory previously reported [Enderlein et al., 1999] which outlines the highly efficient light collection of SAF compared to non-SAF. High quality microscope objective lens with high numerical apertures (NA) could be used to capture more of the non-SAF but this leads to more complicated multiple lens systems with increasing optical losses due to multiple interfaces. This would not suit a POC application. The increase in collection efficiency also indicates that cheaper and less sensitive detectors could be used with this platform.

3.2 Sensitivity assay—biotin-avidin assay

It is accepted convention that assay sensitivity is a functional composite of many contributory factors. Leaving aside operational parameters such as sample variability, heterogeneity and interference, assay sensitivity and detection limits will be predominantly dictated by the instrument performance, reporter signal generation and interpretation and the capture ligand employed. In the case of immuo-based assay platforms the capture ligand is an antibody molecule. These molecules, although highly specific, generally reach maximal affinity in the single digit nanomolar range. Moreover, their relatively bulky nature (150 kDa) imposes diffusional constraints that negatively impact on association rates and can concomitantly limit assay performance when used as labelled reporter ligands. Accordingly, in order to establish a generic measure of both the limit of detection (LOD) and limit of quantitation (LOQ) achievable with the novel paraboloid chip platform, the high affinity biotin-neutravidin (KD = 10−15 M−1) interaction was used as a ligand binding model. The remarkably high affinity of biotin for neutravidin and it’s low molecular weight (244 Da) essentially obviate much of the ligand affinity and diffusional constraints on assay performance and allow a less biased evaluation of the assay platform performance. The 1:1 ratio of biotin to dye molecule allows for a more reliable interpretation of the instrument LOD/LOQ values. Essentialy, neutravidin was immobilised on each paraboloid surface and the biochip was exposed to fluorescently-labelled biotin over a range of concentrations. An appropriate range for the assay was established via several preliminary experiments examining numerous concentrations of the biotin conjugate. The final assay consisted of 6 identical runs performed in duplicate over 3 consecutive days. Each run employed 8 chips, consisting of 7 concentrations of Atto 647-labelled Biotin spiked into 0.5% (w/v) milk-PBST. A zero control consisting of un-spiked buffer was also included in each run. Inter-assay variation was calculated for the assay with coefficient of variation (CV) values ranging from 0.69–13.16%. Figure 6 shows the calibration curve generated by plotting the fluorescence vs the biotin concentration. Both the LOD (zero +5 SD) and LOQ (zero +10 SD) values were calculated from the 4-parameter equation as 4.52 × 1010 and 6.3 × 1010 dye molecules/mL, respectively. This is one parameter to use when determining the absolute analytical sensitivity of an assay platform [Z. Foldes-Papp et al., 2001].
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Fig. 6

Calibration curve showing LOD for biotin-neutravidin assay

3.3 Clinical utility—CRP assay

To demonstrate the clinical applicability of this novel platform, an assay for the detection of CRP, a well-documented inflammatory biomarker, was performed on the paraboloid array chip. A fluorescent sandwich-based format was employed for the detection of several concentrations of CRP spiked into CRP-depleted human serum. It was possible to deploy the in-house generated CRP-specific polyclonal antibody as both the capture and detection component in the sandwich assay due to the plurality of epitopes betrayed by the pentameric CRP molecule. Initially, several coating concentrations of the purified anti-CRP pAb (capture antibody) ranging from 50 to 300 μg/mL were investigated in order to determine the concentration at which saturation occurred. Comparable results were observed for both the 200 μg/mL and 300 μg/mL coating concentrations, indicating saturation of the chip surface. Hence, the lower capture antibody coating concentration of 200 μg/mL was utilised for the assay. The assay comprised a total of 9 identical runs performed in triplicate over 3 consecutive days. A set of 7 CRP concentrations ranging from 10 μg/mL to 0.01 μg/mL, in addition to a ‘zero’ blank represented by unspiked serum, was also incorporated into each run, giving a total of 8 chips per analysis run. The resultant raw data were analysed and inter-day CV values ranging from 2.58–6.73% were observed. A more comprehensive examination of the data was performed using the BIAevaluation data evaluation package (GE Healthcare Bio-Sciences AB, Sweden). The package was used to construct a 4-parameter calibration curve, whereby the F/F0 values were plotted against the CRP concentrations (Fig. 7). This curve was then used to extrapolate theoretical output values for CRP based on the 4-parameter equation. The output recovery values demonstrated a good correlation within the assay. The LOD and LOQ values were calculated from the 4-parameter equation as 7.6 ng/mL and 18 ng/mL, respectively. Notwithstanding that these values are substantially lower than the required actual clinical cut-off levels for CRP, this model assay clearly demonstrates proof of the principle detection of a clinically relevant biomarker analyte in a clinically representative sample matrix using a novel biochip platform.
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Fig. 7

Calibration curve for quantitation of CRP in human serum

3.4 Discrimination from bulk fluorescence

In order to demonstrate the surface-selective capability of the paraboloid elements and, in particular, their ability to discriminate against bulk fluorescence in the analyte solution, the central mask of four of the annular rings was removed. Reaction vessels made from PMMA were adhered to the top of the chip with pressure sensitive adhesive (PSA), with one vessel above each paraboloid element. These were then filled with a solution of Dylight-647-labelled anti-CRP antibody and analysed on the reader. Figure 8 shows the resulting image. Rings 3, 5, 6, 8 and 9 represent fluorescence from paraboloids with annular rings in place. Excitation is due to TIRF so only fluorescence close to the surface is excited. The SAF is collected back through the annular rings, with the rings allowing only fluorescence emitted at the high angles to get through to the detector. In contrast, rings 1, 2, 4 and 7 represent fluorescence from parabola’s without the central mask of the annular ring. Excitation is due both to TIRF and direct excitation from below. The direct excitation excites the bulk fluorescence resulting in increased fluorescence detected that is not specific to the sensor surface. A normal system layout, however, would not allow significant quantities of bulk signal to reach the detector, and as such, the paraboloid chip could measure analyte with the bulk fluorophore solution in situ. As exclusive collection of SAF strictly confines the detection volume to the surface, this would allow measurements to take place with real sample (blood, serum) above the sensor surface negating the need for additional wash steps.
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Fig. 8

Central masks from four annular rings a (1,2,4, & 7) removed to demonstrate the background discrimination

4 Conclusion

We have described a low-cost novel biochip platform consisting of an array of integrated SAF collection elements. The demonstrated high collection efficiency and background discrimination, together with the simplicity, low cost and mass-producibility of the system, makes this biochip platform eminently suitable for POC diagnostic applications. The biochip shows good repeatability and ‘ease-of-use’ using a dedicated biochip reader, both of which allow for low-cost mass production. Each paraboloid can be interrogated individually thereby facilitating multi-analyte sensing when appropriate microfluidics are implemented. The ‘generic sensitivity’ of the system was characterised using a high affinity biotin-neutravidin assay and LOD and LOQ values were found to be 4.52 × 1010 and 6.3 × 1010 dye molecules/mL, respectively. The enhancement of fluorescence capture in a biotin-avidin assay is of the order of 80-100-fold. The system design allows for collection of fluorescence from the surface of the chip while discriminating against fluorescence from the bulk thereby negating the requirement for a wash step. The biochip was validated by measuring the inflammatory marker CRP over the clinical range in serum, giving a LOD of 7.6 ng/mL. Low cost fabrication, dedicated optical interrogation instrumentation and an antibody-compatible surface, allied with the clear amenability for incorporation of a microfluidic sample delivery component, indicate that this device offers a very real alternative to traditional diagnostic assay platforms and has significant potential for application in a POC setting.

Acknowledgments

This material is based upon works supported by the Science Foundation Ireland under Grant No. 05/CE3/B754

Copyright information

© Springer Science+Business Media, LLC 2011