For the first time, we present a simple and robust optical concept to enable precise and sensitive read-out of colorimetric assays in flat lab-on-a-chip devices. The optical guidance of the probe beam through an incorporated measurement chamber to the detector is based on the total internal reflection at V-grooves in the polymer chip. This way, the optical path length through the flat measurement chamber and thus the performance of the measurements are massively enhanced compared to direct (perpendicular) beam incidence. This is demonstrated by a chip-based, colorimetric glucose-assay on serum. Outstanding features are an excellent reproducibility (CV= 1.91 %), a competitive lower limit of detection (cmin = 124 μM), and a high degree of linearity (R2 = 0.998) within a working range extending over nearly three orders of magnitude

Ducrée, Jens

Geipel, A.

Grumann, M.

Kohn, C.

Moser, I.

Riegger, L.

Steigert, J.

Urban, G.

Zengerle, Roland

English

DCU Online Research Access Service

OPTICAL BEAM GUIDANCE IN MONOLITHIC POLYMER CHIPS FOR MINIATURIZED COLORIMETRIC ASSAYS IMTEK, University of Freiburg, GeorgeslKoehler-Allee 106, D-791 I O  Freiburg, Germany 'Lab for MEMS Applications 3 ~ a b  for Sensors 2~~~ ABSTRACT For the first time, we present a simple and robust optical con- cept to enable precise and serisitive read-out of colorimetric assays in flat lab-on-a-chip devices. The optical guidance of the probe beam through an incorporated measurement cham-. ber tothe detector is based on the total internal reflection at V- grooves in the polymer chip. This way, the optical path length through the flat measurement chamber and thus the perfor- mance of the measurements are massively enhanced compared to direct (perpendicular) beam incidence. This is demonstrated by a chip-based, colorimetric glucose-assay on serum. Out- standing features are an excellent reproducibility (CV= 1.91 %), a competitive lower limit of detection (cmi,., = 124 pM), and a high degree of linearity (R2 = 0.998) within a working range extending over nearly three orders of magnitude. 1. INTRODUCTION Colorimetric absorption is, apart from electrochemical detec- tion [l], one of the most commonly used modes of detection for assays on miniaturized lab-on-a-chip systems. This read- out method utilizes the specific absorption of a probe beam as a measure for the initial concentration of a biochemical target. For these miniaturized systems, a major drawback is the rela- tively short optical path length labs through the measurement chamber which is limited by the physical dimensions of the system..Several approaches to guide an optical beam on-chip and thus to optimize the sensitivity of the system are related to embedded optical fibers [2], integrated' microlenses 131 or waveguides [4,5]. I .  Since these approaches require rather complex designs and manufacturing processes, we present .a novel concept to ex- tend the optical path length by total internal reflection (TIR) at integrated V-grooves. This way, the optical path through the measurement chamber becomes independent of the thickness of the chip and is extended to enhance the performance of the sensor. Additionally, the monolithic devices can be manufac- tured by standard micromachining and do not require on-chip alignment of optical elements. 2. SETUP We use a micromachined polymer disk (COC) with the size of a common compact disk (CD) as the substrate 161. It features fluidic and optical elements to perform colorimetric assays. The fluidic elements provide ports for sample and reagent up- take which are connected to a combined mixing and measure- ment chamber (Fig. 1). The liquid transport is controlled by centrihgai forces F, which are generated by spinning of the disk [7j. -\ _______I_ - --- measurement I COC: n, 1.5 Figure 1. Schematic o j a  microjluidic disk which combines fluidic elements to conduct a colorimetric assay with optical elements for on-chip optical beam guidance by total intemal rejection VIR). Inlet ports to load the sampIe and the reagents are connected to the measurement chamber where mixing of the Iiquids and the read-out of the assay are con- ducted. Next to the .measurement chambe6 V-grooves are embedded to couple the beam into the disk-plane. By spin- ning the disk, fhe sample and reagents are trunsportedfiom their respective inlets into the meusurement chamber As optical guidance structure, triangular V-grooves are em- bedded next to the measurement chamber at the reverse side of the chip. When the beam has entered the polymer chip, it is de- flected via total internal reflection (TIR) at the side face of the V-groove. To ensure complete reflection, the angle of inci- dence a h a s  to exceed the critical angle cy, for TIR according to the law of Snellius (Fig. 2). With n, = 1.5 and n,  = 1 as the refractive inijices of the applied polymer substrate (COC) and the surrounding air, respectively, the critical angle of total in- ternal reflection amounts to cy, = 41". 0-7803-8732-5/05/$20.00 0 2005 EEE. ,108 Authorized licensed use limited to: DUBLIN CITY UNIVERSITY. Downloaded on July 12,2010 at 13:14:39 UTC from IEEE Xplore.  Restrictions apply. 1 I I Fsneliius: 0" 45" 90' Figure 2. Beam-guidance by total internal rejlection (TIR). Governed by the law of Snellius, the critical angle ~2. ,  for TIR at the interfiace of the applied polymer with n, = 1.5 and air with n ,  = 1 is derived to be r y ~  =41 4 Thus, an opti- cal beam with an incidence aiigle c y =  45" is totally reflected perpendiczdar to the incidence. The optical beam of a standard laser diode [SI is directed at perpendicular incidence on the flat upper side of the chip. Af- ter deflection into the chip-plane, the probe beam with an ini- tial intensity 10 is attenuated in the measurement chamber and then reflected at another V-groove towards a collimation-op- tics [9]  positioned above the chip which focuses the beam onto the aperture of a fiber-probed micro-spectrophotometer [ 101 (Fig. 3) where the attenuated intensity 1 is measured. With this setup, an optical path length of lab, = 10 mm through the mea- surement chamber has been realized, even though the height of the chamber amounts to 1 mm, only. Governed by the law of Beer-Lambert I A = -In- = c .  c .  labs,  10 with €as the extinction coefficient and c as the target concen- tration, the absorbance A increases with labS, thus the perfor- mance of the sensor is enhanced. 3. COLORIMETRIC GLUCOSE-ASSAY In clinical diagnostics, a multitude of metabolic parameters are analyzed from whole blood or serum. Most of the methods involve several steps including the detection reaction. To quantify the sensor performance, we present a blood glucose determination which is one of the most important assays in clinical diagnostics. The chosen peroxidase based detection method can be used also for the determination of other meta- bolic parameters like lactate as well as immuno-assays. The assay is conducted via a colorimetric, two-step reaction scheme (Fig. 4). As a first step, hydrogen peroxide (H202) is produced by the catalytic oxidation of glucose with glucose oxidase (GOD). labs=1 mm Figure 3. OpticaI elemenrs to perfom TIR on-chip. Next to the measurement chambe6 integruted V-grooves deflect the beam into the disk-plane by TlR. Afrer passing the measure- ment chambel; the attenuatedprobe beam is measured by a detector positioned nbove the disk. This concept signif- cantly extends the optical path Iength of the beam through the frat poIymsr disk to lobs = 10 mm compared to direct incidence (labs = 1 mm), thus sensitive colorimetric assuys can be pe ?$ormed. Subsequently, ABTS(red) is oxidized to ABTS(ox) by I3202 in a POD catalyzed reaction. For the read-out of the assay, a laser diode with an emission wavelength of /Ipeak = 650 nm is ap- plied, although the extinction maximum of the ABTS(ox) dye is located at R = 420 nm. However, there is sufficient attenua- tion for sensitive detection at the selected laser wavelength. GOD 6-Gtuconolactone + H202 A Glucose + O2 2 ABTS(red) + H20z 'OD + 2 ABTS(ox) + 2 H20 e Maximum 6 Difference spectrum : @ A = 420 nm for a glucoseassay ro 1.0: with c = 13.75 mM 5 0.8- i 0.6- B Y CI 1 .- 0 0 E 0.4- F 0.2- = 0.0 L3  & ' & ' & % J ' & '  Wavelength 1 (nm) Figure 4. Colorimetric determination of the glucose con- centration via a fwo-step enzymatic reaction. (A) First, hydrogen peroxide (HZO2) is generated in the catalyzed oxi- dation of glucose by glucose oxidase (GOD). Subsequent&, the oxidation of the ABTS(red) bye by HtO, to ABTS(ox) is catalyzed by peroxidase (POD). {B) Though the dflerence spectrum of AETS(ox) features an extinction maximum at A 420 nm, the applied laser with a wavelength of = 650 nm still experiences a suficient attenuation jor  a sensitive assay read-out. 109 Authorized licensed use limited to: DUBLIN CITY UNIVERSITY. Downloaded on July 12,2010 at 13:14:39 UTC from IEEE Xplore.  Restrictions apply. To perform the assay, a droplet of sample (500 nl) is dispensed into the chip, diluted with PBS-buffer (30.2 pl) and mixed with the reagents (69.3 pl), namely GOD (40 units/ml), POD (4 unitdml), and ABTS(red) (2 mM), in the measurement chamber. To enhance the diffusion-limited speed of the reaction, we transferred the assay on our previously developed ‘lab-on-a- disk’ platform [11,12] (Fig. 5) .  By frequent reversal of the sense of rotation (’shake-mode’) for a period of 2 minutes, in- ertially induced advection accelerates mixing from 6.5 minutes for mere diffusion to 5.1 seconds [13]. t =os t =0.3s t = l . O s  t =I 0.0s ---U reference: r = 6.5 min o’81r 0.6 0.2 shake mode mixing: T = 5.1 s 0.41 0 0  I 5 10 15 20 Mixing time t (s) Figure 5. Accelerated mixing by frequent reversal of the sense of rotation (khuke-mode ’). Upon acceleration and deceleration, inerfial effects induce advective currents which reduce the difision length. This wuy, the characteris- tic mixing time t is reduced from 6.5 min for mere difision to 5.1 s, only. These measurements are calibrated by a series of experiments with horse serum at known glucose concentrations (Fig. 6). The data-points display an absorption characteristics comply- ing with the law of Beer-Lambert (equ. 1). We obtain a CVof 1.91 %, c ~ n  = 124 pM its the lower limit of detection, and a highly linear relation between the glucose concentration and the optical signal (R2 = 0.998). Additionally, the on-chip con- cept of guiding an optical beam with an optical path length of labs = 10 mm was compared with a rather conventional align- ment (i.e.; transmission) with labs = 1 mm. It turns out that, as predicted by equation 1 , the slope AAlAc and thus the resolu- tion follows the ratio of the absorption lengths (Fig. 7) and is improved by one order of magnitude. 2.5 2 5 1.5 c : 0 I CI g 1  (D 9 0.5 0 0 5 10 15 20 25 Glucose concentration c (mM) Figwe 6. Results of glucose determination: a sample of horse serum with an initial gIucose concentration c = 4.0 mM is spiked to extend the concentration up to 25 m M  mth  a CY of 1.91 %, a lower detection limit of c,, = 124 pM and a Iinearity of R2 = 0.998, our setup allows direct measuvements at high precision. 2.5 - 2  z : 1.5 S I  B 0.5 0 q C 0 U 0 5 10 15 20 25 Glucose concentration c (mM) Figure 7. Improvement of the sensor performance by an enhanced optical path length lobs. The slope of the charac- teristic absorption curve AA/Ac, which is U decisive impact facfor for  the resolution, is improved by a factor of 10 com- pared to transmission by pevendicular incidence of the beam at theflat side of the device without deflection into the chip-plane. 4. CONCLUSION We realized a versatile optical method for high-performance read-out of colorimetric assays by enhancing the optical path length via total internal reflection. The simple and rugged con- cept is implemented in a modular setup comprising an optical beam source, a detector, and an exchangeable polymer sub- strate containing a flat measurement chamber and monolithi- cally integrated optical guidance structures. The microfluidic chip can thus be readily fabricated by standard polymer micro- machining techniques and can be recommended for other for- mats of absorbance assays including ELISAs. 110 Authorized licensed use limited to: DUBLIN CITY UNIVERSITY. Downloaded on July 12,2010 at 13:14:39 UTC from IEEE Xplore.  Restrictions apply. ACKNOWLEDGEMENTS The authors are grateful to the partial support by the Ministry of Science, Research and the Arts of the German federal state of Baden-Wuerttemberg (contract 24-720.43 1-1 -712) and the good cooperation with HSG-IMIT and Jobst-Technologies. REFERENCES [ 1 J I. Moser, G Jobst, and G. Urban, Biosensor Arrays for Si- multaneous Measurement of Glucose, Lactate, Glutamate, and Glutamine, Biosensors & Bioelectronics, 17,297-302,2002. [2j Z. Liang, N. Chiem, G. Ocvirk, T. Tang, K. Fluri, and D. .Ted Harrsion, Microfabrication of a Planar Absorbance and Fluorescence Cell for Integrated Capillary Electrophoresis Devices, Anal, Chem., 68, 104@-1046, 1996. [3] J. Roulet, R. Volkel, H. Herzig, E. Verpoorte, and N. de Rooij, Fabrication of MultiIayer Systems Combining Microf- luidic and Microoptical Elements for Fluorescence Detection, Journal of Microelectromechanical Systems, 10, 482-491, 2001. [4] N. Petersen, K. Mogensen, and J. Kutter, Performance of an in-plane Detection Cell with Integrated Waveguides for UVNIS Absorbance Measurements on Microfluidic Separa- tion Devices, Electrophoresis, 23,3528-3536,2002. [SI B. Splawn and F. Lytle, On-chip Absorption Measure- ments using Integrated Waveguides, Anal. Bioanal. Chem., 373,5 19-525,2002, [6] Jobst-Technologies GmbH, Germany, -.jobst-technol- ogies.com. [7] M. Madou, Y. Lu, S .  Lai, J. Lee, and S .  Daunert, A Centrif- ugal Microfluidic Platform - A Comparison, Proc. pTAS 2000 conference, A. van den Berg and P. Bergveld (editors), pp. 565 - 570, Kluwer Academic Publisher, 2000. [XI Laser diode LDM650IlL.J fixfocus, Roithner Lasertech- nik, Austria, www.roithner-laser.com. [SI Fiber collimation package F220SMA-A, Thorlabs GmbH, Germany, www.thorlabs.com. [ 101 WNIS-Microspectrometer, STEAG microParts GmbH, Germany, www.microparts.de. [ I  I] S .  Lai, S .  Wang, J. Luo, S. Yang and M. Madou, Design of a Compact Disk-like Microfluidic Platform for Enzyme- linked Immunosorbent Assay, Anal. Chem., 76, 1832-1837, 2004. [12j I. Badr, R. Johnson, M. Madou, and L. Bachas, Fluores- cent Ion-selective Optode Membranes Incorporated onto a Centrifugal Microfluidics Platform, Anal. Chem:, 74, 5569 - 5575,2004. [13] M. Grumann, A. Geipel, L. Riegger, R. Zengerle and J. Ducree, Magneto-Hydrodynamic Micromixing for Centrih- gal Lab-on-a-Disk Platforms, Proc. bTAS conference 2004, 593-595,2004. 111 Authorized licensed use limited to: DUBLIN CITY UNIVERSITY. Downloaded on July 12,2010 at 13:14:39 UTC from IEEE Xplore.  Restrictions apply. 

A Centrifugal Microfluidic Platform - A Comparison,

Authorized licensed use limited to: DUBLIN CITY UNIVERSITY.

Biosensor Arrays for Simultaneous Measurement of Glucose, Lactate,

Design of a Compact Disk-like Microfluidic Platform for Enzymelinked Immunosorbent Assay,

Fabrication of MultiIayer Systems Combining Microfluidic and Microoptical Elements for Fluorescence Detection,

Fluorescent Ion-selective Optode Membranes Incorporated onto a Centrifugal Microfluidics Platform,

Magneto-Hydrodynamic Micromixing for Centrihgal Lab-on-a-Disk Platforms,

Microfabrication of a Planar Absorbance and Fluorescence Cell for Integrated Capillary Electrophoresis Devices,

On-chip Absorption Measurements using Integrated Waveguides,

Performance of an in-plane Detection Cell with Integrated Waveguides for UVNIS Absorbance Measurements on Microfluidic Separation Devices, Electrophoresis,

XI Laser diode LDM650IlL.J fixfocus,

Optical beam guidance in monolithic polymer chips for miniaturized colorimetric assays

Optical beam guidance in monolithic polymer chips for miniaturized colorimetric assays

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