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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 18, Iss. 18 — Aug. 30, 2010
  • pp: 19120–19128
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Low-cost label-free biosensors using photonic crystals embedded between crossed polarizers

Yousef Nazirizadeh, Uwe Bog, Sylwia Sekula, Timo Mappes, Uli Lemmer, and Martina Gerken  »View Author Affiliations


Optics Express, Vol. 18, Issue 18, pp. 19120-19128 (2010)
http://dx.doi.org/10.1364/OE.18.019120


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Abstract

There is a strong need for low-cost biosensors to enable rapid, on-site analysis of biological, biomedical, or chemical substances. We propose a platform for label-free optical biosensors based on applying the analyte onto a surface-functionalized photonic crystal slab and performing a transmission measurement with two crossed polarization filters. This dark-field approach allows for efficient background suppression as only the photonic crystal guided-mode resonances interacting with the functionalized surface experience significant polarization rotation. We present a compact biosensor demonstrator using a low-cost light emitting diode and a simple photodiode capable of detecting the binding kinetics of a 2.5 nM solution of the protein streptavidin on a biotin-functionalized photonic crystal surface.

© 2010 OSA

1. Introduction

Biosensors are devices that use biological materials to detect other biological, biomedical, or chemical substances [‎1

1. J. N. Anker, W. P. Hall, O. Lyandres, N. C. Shah, J. Zhao, and R. P. Van Duyne, “Biosensing with plasmonic nanosensors,” Nat. Mater. 7(6), 442–453 (2008). [CrossRef] [PubMed]

-8

8. F. Vollmer, D. Braun, A. Libchaber, M. Khoshsima, I. Teraoka, and S. Arnold, “Protein detection by optical shift of a resonant microcavity,” Appl. Phys. Lett. 80(21), 4057–4059 (2002). [CrossRef]

]. In particular, these devices are used to measure protein-protein interactions, binding affinities, and kinetic processes. These biosensors are applied for the determination of active concentrations, for screening, and for the characterization of biological substances. All these tests are critical processes for, e.g., biological research and drug discovery. Today’s established optical label-free methods are based on surface plasmon resonances (SPR) [‎1

1. J. N. Anker, W. P. Hall, O. Lyandres, N. C. Shah, J. Zhao, and R. P. Van Duyne, “Biosensing with plasmonic nanosensors,” Nat. Mater. 7(6), 442–453 (2008). [CrossRef] [PubMed]

] or optical modes in resonator structures with high quality factors [‎5

5. A. M. Armani, R. P. Kulkarni, S. E. Fraser, R. C. Flagan, and K. J. Vahala, “Label-free, single-molecule detection with optical microcavities,” Science 317(5839), 783–787 (2007). [CrossRef] [PubMed]

-‎8

8. F. Vollmer, D. Braun, A. Libchaber, M. Khoshsima, I. Teraoka, and S. Arnold, “Protein detection by optical shift of a resonant microcavity,” Appl. Phys. Lett. 80(21), 4057–4059 (2002). [CrossRef]

]. To detect a given analyte by these methods the respective resonance is spectrally analyzed; a procedure that often requires spectrometers and computational resources. Due to the high acquisition costs of such devices their application is limited. On the other hand, compact biosensors are promising tools for medical diagnostics. For instance, detection of biomarkers has much improved the diagnostics of diseases such as cancer [‎9

9. G. Zheng, F. Patolsky, Y. Cui, W. U. Wang, and C. M. Lieber, “Multiplexed electrical detection of cancer markers with nanowire sensor arrays,” Nat. Biotechnol. 23(10), 1294–1301 (2005). [CrossRef] [PubMed]

]. In this work we present a low-cost and compact technology platform for label-free biosensors based on surface-functionalized photonic crystal slabs and efficient background suppression.

2. Guided-mode resonance shift determination via intensity measurements

Using a PCS as transducer for biosensor applications, a key process is the determination of the spectral position shift of GMRs. To solve this issue in a cost-efficient and compact way, we propose to use a light source possessing a rising or a falling edge in the spectral region of the GMRs. The convolution of the shifted GMR with the spectrum of the light source results in a function of GMR’s spectral shift versus the intensity of the transmission. This function shows a pure positive slope, when GMRs overlap with the rising edge of the light source, and a negative slope for GMRs overlapping with the sloping edge of the light source. Therefore, a simple intensity measurement can replace spectral analysis of GMRs.

These considerations are summarized in an example depicted in Fig. 2(a)
Fig. 2 (a) Basic concept of the conversion of GMR shift into intensity change using an LED as the light source. The convolution of the LED spectrum with the GMR results in a function of the spectral shift of GMR versus the intensity of the transmission. (b) Schematic of a compact and low-cost biosensor for affinity measurements in real time. Here, an LED is applied as the light source, while a photo diode is used for the intensity detection.
. Here the spectrally limited light source is chosen to be a standard LED, for which the GMR overlaps only with its falling edge. A refractive index change on the surface of the PCS would result in a shift of the GMR. The intensity of the light source is reduced at the new spectral position of the GMR and therefore the transmitted intensity is decreased. A simple setup, as shown in Fig. 2(b) contains all required elements to realize the desired device. Here, solely a photodiode is utilized to detect the intensity of the transmission.

3. Bulk refractive index measurements

A series of water-sucrose (C12H22O11) dilutions is prepared, which is used to cover the PCS surface. In Fig. 3(a)
Fig. 3 (a) Transmission measurements with tuned refractive index and an LED as the light source. An intensity drop of up to 75% is observed, which stands in relation to the GMR shift. (b) Integrated intensity as deduced from spectra versus refractive index of the surrounded liquid obtained by integrating spectra obtained in Fig. 3(a). The decreasing intensity is due to the GMR shift, which is a function of the refractive index. As expected the intensity drop follows the sloping edge of the LED spectrum.
11 measurements with sucrose concentrations from 0 g/ml to 1 g/ml with 0.1 g/ml steps are shown. Comparing these transmission measurements with results obtained in Fig. 1(b), we observe differences in mode intensity and spectral position. This is a result of the limited LED spectrum and its radiation characteristics. The short interaction length of the light with the solution and the low sucrose concentrations allow us to neglect the specific rotation of sucrose, which is a polarization rotation of light passing the solution.

Due to the tuned refractive index, GMRs show an average spectral shift of about 10 nm. Moreover, we observe as expected an intensity drop for all modes and a maximum change of 75% for individual modes.

The integration of the spectra over all GMRs yields intensity values comparable with those expected for a photodiode measurement. In Fig. 3(b) this intensity curve is plotted versus the associated refractive index. As expected, the intensity of transmitted GMRs follows the sloping edge of the LED spectrum and drops by more than 45%.

4. Compact demonstrator

To demonstrate the potential of this method to allow for compact and cost-efficient systems, we designed and realized a demonstrator. The setup of the demonstrator is as simple as depicted in Fig. 2(b). To detect the transmitted intensity, which is a function of the GMR shift, we use a silicon photodiode. The LED and the photodiode exhibit integrated epoxy optics. On the LED side this optics partly parallelizes the emitted light and directs it towards the detector. On the detector side the epoxy optic focuses the transmitted light into the detecting area.

The flow-cell (Fig. 4(a)
Fig. 4 (a) Photograph of the flow-cell during volume change with an ink-water dilution. The flow-cell consists of an o-ring squeezed between two glass substrates (one with the PCS on its surface). The liquid supply is realized using a butterfly cannula, which is pierced into the o-ring. The capacity of the flow-cell depends on the diameter and the thickness of the o-ring and was in this case approximately 200 µl. (b) Photograph of the assembled biosensor demonstrator.
) consists of an elastic o-ring squeezed between a glass substrate and the PCS, which is fabricated on a glass substrate as well, using fold back clips. A liquid supply containing the analyte is realized using a butterfly cannula, which is pierced into the o-ring. Similarly for the outflow also a butterfly cannula is used. The pumping of the liquid is performed by manual operation of a syringe through the supply cannula, while the outflow cannula is opened. The liquid capacity of the flow-cell depends on the diameter and the thickness of the o-ring. In the present case the liquid volume was approximately 200 µl. For bulk refractive index measurements a complete liquid exchange of the flow-cell is of high impact. For this flow-cell 3 ml fluid sufficed for this purpose.

To combine all components of the demonstrator in a compact way and guarantee correct adjustment, a polymer frame was designed by CAD software and fabricated using a 3D plotter. The 3D plotter uses a polymeric material for the fabrication and provides a resolution of about 0.1 mm. A photograph of the assembled biosensor demonstrator is shown in Fig. 4(b).

As the resonance shift determination is converted into an intensity measurement, a stable luminous flux of the light source is an essential issue. For this purpose we use a current source, which delivers a constant current independent of the LED voltage. The photodiode, on the other hand, is driven in a short circuit configuration with a current-to-voltage converter. The output voltage is analyzed and recorded using a data acquisition device, which was connected to a computer.

Again a bulk refractive index study is performed to estimate the detection limit of the demonstrator. We prepared three solutions with refractive indices close to each other, using water-isopropanol dilutions. With an isopropanol concentration of 0%, 0.5% and 1%, we achieved tuning of the refractive index in three steps with a Δn = 2.25 10−4 (with a refractive index of 1.333 @20°C for water and 1.378 @20° C for isopropanol).

We pumped these solutions into the flow-cell in two experiments. First, we alternate between pure water and 1% isopropanol dilution and exchange the whole volume of the flow-cell in periods with duration of 60 s. In a second experiment we alternate between water and 0.5% isopropanol. The injection into the flow-cell is performed with a syringe with a capacity of 3 ml and has a duration of about 10 s. Both results are plotted in Fig. 5
Fig. 5 Determination of the detection limit of the demonstrator, utilizing bulk refractive index tests. A 0.5% isopropanol-water dilution could be clearly differentiated from pure water. Hence, a bulk refractive index detection limit of Δn = 2.25 10−4 is obtained.
, which is the output signal in voltage as a function of time. We observed three signal level, namely for pure water, for 0.5% and 1% isopropanol dilution. These signal levels are equally spaced with a voltage relative difference of about 0.13%.

In addition, we observe signal peaks at every injection procedure (Fig. 5). We believe that this behaviour is caused due to the overpressure in the flow-cell during the injection procedure. These peaks are either due to refractive index changes caused by changed pressure or, more likely, because of displacement of the PCS relative to the light source.

5. Real-time label-free detection of streptavidin binding to biotin

To validate the optoelectronic sensor application as a biomolecule detector, we studied a key-lock system composed of streptavidin and biotin, two molecules that have great affinities to each other [‎16

16. T. Sano and C. R. Cantor, “Intersubunit contacts made by tryptophan 120 with biotin are essential for both strong biotin binding and biotin-induced tighter subunit association of streptavidin,” Proc. Natl. Acad. Sci. U.S.A. 92(8), 3180–3184 (1995). [CrossRef] [PubMed]

]. The surface of the PCS was functionalized with biotin, using a compound of biotinylated phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (N-Biotinyl Cap-PE) mixed in different mol% ratios with phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in order to control the amount of the functional biotin-headgroup lipids deposited on the surface. We chose spin-coating as the method to apply biotinylated phospholipids on the PCS surface due to its simplicity of application and possibility to control the coating conditions. We used the flow-cell in order to apply streptavidin solution in phosphate buffered saline (PBS) (concentrations varying from 50 nMol to 2.5 nMol) onto the biotin-functionalized PCS surface. Figure 6
Fig. 6 Spectral analysis of the second resonance with a spectrometer and an LED as light source during the streptavidin coupling process. We observe a resonance shift of 0.5 nm as well as an intensity decrease of the transmission following the falling edge of the LED spectrum.
shows the time-dependent transmission analyzed as a control experiment with a spectrometer using an LED as light source. In this experiment, we injected 50 nMol of streptavidin into the flow-cell and characterized the second resonance of the PCS over a total time of 1,000 seconds. We observed a resonance shift of Δλ = 0.5 nm as well as an intensity decrease of the transmission, which follows the falling edge of the LED spectrum.

We have applied the presented demonstrator and have performed affinity measurements in real time. Figure 7(a)
Fig. 7 (a) Relative voltage reduction using demonstrator of (Fig. 4a) as a function of time for two different composition ratios of N-Biotinyl Cap-PE admixed in DOPC. Signal saturation is reached faster with a 10 mol% of N-Biotinyl Cap-PE content in DOPC. (b) The influence of streptavidin concentration on the signal. Lowering the streptavidin concentration, the saturation level decreases. 2.5nMol streptavidin binding to N-Biotinyl Cap-PE functionalized surface was clearly detectable.
shows the effect of the ratio of biotin-DOPC mixtures on the signal. The green and the red curves show the relative signal response to 25 nMol streptavidin for the surface functionalized with 10 mol% and 4 mol% biotinylated DOPC, respectively. As expected, the streptavidin-biotin binding process was accelerated at a higher concentration of biotin. Furthermore, we observe saturation of the signal after 1,000 s. for the surface functionalized with 10 mol% biotinylated DOPC. This indicates that all the streptavidin molecules in the flow-cell are bound to the surface. The inhomogeneous rise of the signal is manifested by the non-homogeneous functionalization, which might be caused by imperfect spin-coating. Using 10 mol% biotinylated DOPC as functionalization, we reduced the concentration of streptavidin to 2.5 nMol and still observe a relative signal reduction of about 0.4% (Fig. 7(b)).

6. Conclusions

In conclusion, we have introduced a novel easy-to-implement technology platform for biosensors based on functionalized PCS in combination with crossed polarization filters. Based on the proposed platform, we presented a biosensor demonstrator that uses an LED as light source and a photodiode as detector to perform label-free molecular affinity measurements in real time. We detected a 2.5 nMol streptavidin solution, which is a relevant concentration in life sciences.

Acknowledgments

We thank U. Geyer of the Light Technology Institute at the Karlsruhe Institute of Technology (KIT) for providing the sample photonic crystal slabs. Furthermore, we acknowledge support by the German Federal Ministry for Education and Research BMBF within the NanoFutur program (Project No. 03X5514). U. Bog acknowledges support by the Karlsruhe School of Optics & Photonics (KSOP).

References and links

1.

J. N. Anker, W. P. Hall, O. Lyandres, N. C. Shah, J. Zhao, and R. P. Van Duyne, “Biosensing with plasmonic nanosensors,” Nat. Mater. 7(6), 442–453 (2008). [CrossRef] [PubMed]

2.

Y. Cui, Q. Wei, H. Park, and C. M. Lieber, “Nanowire nanosensors for highly sensitive and selective detection of biological and chemical species,” Science 293(5533), 1289–1292 (2001). [CrossRef] [PubMed]

3.

B. Polisky, R. Jenison, S. Yang, and A. Haeberli, “Interference-based detection of nucleic acid targets on optically coated silicon,” Nat. Biotechnol. 19(1), 62–65 (2001). [CrossRef] [PubMed]

4.

V. S. Y. Lin, K. Motesharei, K. P. S. Dancil, M. J. Sailor, and M. R. Ghadiri, “A porous silicon-based optical interferometric biosensor,” Science 278(5339), 840–843 (1997). [CrossRef] [PubMed]

5.

A. M. Armani, R. P. Kulkarni, S. E. Fraser, R. C. Flagan, and K. J. Vahala, “Label-free, single-molecule detection with optical microcavities,” Science 317(5839), 783–787 (2007). [CrossRef] [PubMed]

6.

E. Chow, A. Grot, L. W. Mirkarimi, M. Sigalas, and G. Girolami, “Ultracompact biochemical sensor built with two-dimensional photonic crystal microcavity,” Opt. Lett. 29(10), 1093–1095 (2004). [CrossRef] [PubMed]

7.

B. T. Cunningham, P. Li, S. Schulz, B. Lin, C. Baird, J. Gerstenmaier, C. Genick, F. Wang, E. Fine, and L. Laing, “Label-free assays on the BIND system,” J. Biomol. Screen. 9(6), 481–490 (2004). [CrossRef] [PubMed]

8.

F. Vollmer, D. Braun, A. Libchaber, M. Khoshsima, I. Teraoka, and S. Arnold, “Protein detection by optical shift of a resonant microcavity,” Appl. Phys. Lett. 80(21), 4057–4059 (2002). [CrossRef]

9.

G. Zheng, F. Patolsky, Y. Cui, W. U. Wang, and C. M. Lieber, “Multiplexed electrical detection of cancer markers with nanowire sensor arrays,” Nat. Biotechnol. 23(10), 1294–1301 (2005). [CrossRef] [PubMed]

10.

S. Fan and J. D. Joannopoulos, “Analysis of guided resonances in photonic crystal slabs,” Phys. Rev. B 65(23), 235112 (2002). [CrossRef]

11.

Y. Nazirizadeh, J. G. Müller, U. Geyer, D. Schelle, E.-B. Kley, A. Tünnermann, U. Lemmer, and M. Gerken, “Optical characterization of photonic crystal slabs using orthogonally oriented polarization filters,” Opt. Express 16(10), 7153–7160 (2008). [CrossRef] [PubMed]

12.

Y. Nazirizadeh, U. Lemmer, and M. Gerken, “Experimental quality factor determination of guided-mode resonances in photonic crystal slabs,” Appl. Phys. Lett. 93(26), 261110 (2008). [CrossRef]

13.

Y. Nazirizadeh, J. G. Müller, U. Geyer, U. Lemmer, and M. Gerken, “Direct observation of photonic modes in photonic crystal slabs,” in Proceedings of International Conference on Transparent Optical Networks (Academic, 2008), pp. 72–75.

14.

U. Geyer, J. Hauss, B. Riedel, S. Gleiss, U. Lemmer, and M. Gerken, “Large-scale patterning of indium tin oxide electrodes for guided mode extraction from organic light-emitting diodes,” J. Appl. Phys. 104(9), 093111 (2008). [CrossRef]

15.

L. Shi, P. Pottier, Y. A. Peter, and M. Skorobogatiy, “Guided-mode resonance photonic crystal slab sensors based on bead monolayer geometry,” Opt. Express 16(22), 17962–17971 (2008). [CrossRef] [PubMed]

16.

T. Sano and C. R. Cantor, “Intersubunit contacts made by tryptophan 120 with biotin are essential for both strong biotin binding and biotin-induced tighter subunit association of streptavidin,” Proc. Natl. Acad. Sci. U.S.A. 92(8), 3180–3184 (1995). [CrossRef] [PubMed]

OCIS Codes
(170.0170) Medical optics and biotechnology : Medical optics and biotechnology
(280.4788) Remote sensing and sensors : Optical sensing and sensors
(050.5298) Diffraction and gratings : Photonic crystals

ToC Category:
Photonic Crystals

History
Original Manuscript: May 27, 2010
Revised Manuscript: August 18, 2010
Manuscript Accepted: August 20, 2010
Published: August 25, 2010

Virtual Issues
Vol. 5, Iss. 13 Virtual Journal for Biomedical Optics

Citation
Yousef Nazirizadeh, Uwe Bog, Sylwia Sekula, Timo Mappes, Uli Lemmer, and Martina Gerken, "Low-cost label-free biosensors using photonic crystals embedded between crossed polarizers," Opt. Express 18, 19120-19128 (2010)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-18-18-19120


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References

  1. J. N. Anker, W. P. Hall, O. Lyandres, N. C. Shah, J. Zhao, and R. P. Van Duyne, “Biosensing with plasmonic nanosensors,” Nat. Mater. 7(6), 442–453 (2008). [CrossRef] [PubMed]
  2. Y. Cui, Q. Wei, H. Park, and C. M. Lieber, “Nanowire nanosensors for highly sensitive and selective detection of biological and chemical species,” Science 293(5533), 1289–1292 (2001). [CrossRef] [PubMed]
  3. B. Polisky, R. Jenison, S. Yang, and A. Haeberli, “Interference-based detection of nucleic acid targets on optically coated silicon,” Nat. Biotechnol. 19(1), 62–65 (2001). [CrossRef] [PubMed]
  4. V. S. Y. Lin, K. Motesharei, K. P. S. Dancil, M. J. Sailor, and M. R. Ghadiri, “A porous silicon-based optical interferometric biosensor,” Science 278(5339), 840–843 (1997). [CrossRef] [PubMed]
  5. A. M. Armani, R. P. Kulkarni, S. E. Fraser, R. C. Flagan, and K. J. Vahala, “Label-free, single-molecule detection with optical microcavities,” Science 317(5839), 783–787 (2007). [CrossRef] [PubMed]
  6. E. Chow, A. Grot, L. W. Mirkarimi, M. Sigalas, and G. Girolami, “Ultracompact biochemical sensor built with two-dimensional photonic crystal microcavity,” Opt. Lett. 29(10), 1093–1095 (2004). [CrossRef] [PubMed]
  7. B. T. Cunningham, P. Li, S. Schulz, B. Lin, C. Baird, J. Gerstenmaier, C. Genick, F. Wang, E. Fine, and L. Laing, “Label-free assays on the BIND system,” J. Biomol. Screen. 9(6), 481–490 (2004). [CrossRef] [PubMed]
  8. F. Vollmer, D. Braun, A. Libchaber, M. Khoshsima, I. Teraoka, and S. Arnold, “Protein detection by optical shift of a resonant microcavity,” Appl. Phys. Lett. 80(21), 4057–4059 (2002). [CrossRef]
  9. G. Zheng, F. Patolsky, Y. Cui, W. U. Wang, and C. M. Lieber, “Multiplexed electrical detection of cancer markers with nanowire sensor arrays,” Nat. Biotechnol. 23(10), 1294–1301 (2005). [CrossRef] [PubMed]
  10. S. Fan and J. D. Joannopoulos, “Analysis of guided resonances in photonic crystal slabs,” Phys. Rev. B 65(23), 235112 (2002). [CrossRef]
  11. Y. Nazirizadeh, J. G. Müller, U. Geyer, D. Schelle, E.-B. Kley, A. Tünnermann, U. Lemmer, and M. Gerken, “Optical characterization of photonic crystal slabs using orthogonally oriented polarization filters,” Opt. Express 16(10), 7153–7160 (2008). [CrossRef] [PubMed]
  12. Y. Nazirizadeh, U. Lemmer, and M. Gerken, “Experimental quality factor determination of guided-mode resonances in photonic crystal slabs,” Appl. Phys. Lett. 93(26), 261110 (2008). [CrossRef]
  13. Y. Nazirizadeh, J. G. Müller, U. Geyer, U. Lemmer, and M. Gerken, “Direct observation of photonic modes in photonic crystal slabs,” in Proceedings of International Conference on Transparent Optical Networks (Academic, 2008), pp. 72–75.
  14. U. Geyer, J. Hauss, B. Riedel, S. Gleiss, U. Lemmer, and M. Gerken, “Large-scale patterning of indium tin oxide electrodes for guided mode extraction from organic light-emitting diodes,” J. Appl. Phys. 104(9), 093111 (2008). [CrossRef]
  15. L. Shi, P. Pottier, Y. A. Peter, and M. Skorobogatiy, “Guided-mode resonance photonic crystal slab sensors based on bead monolayer geometry,” Opt. Express 16(22), 17962–17971 (2008). [CrossRef] [PubMed]
  16. T. Sano and C. R. Cantor, “Intersubunit contacts made by tryptophan 120 with biotin are essential for both strong biotin binding and biotin-induced tighter subunit association of streptavidin,” Proc. Natl. Acad. Sci. U.S.A. 92(8), 3180–3184 (1995). [CrossRef] [PubMed]

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