## Molecular film growth monitoring via reflection microscopy on periodically patterned substrates |

Optics Express, Vol. 21, Issue 4, pp. 4215-4227 (2013)

http://dx.doi.org/10.1364/OE.21.004215

Acrobat PDF (1296 KB)

### Abstract

An optical method is presented for in the situ monitoring of biomolecular films via reflection microscopy on patterned substrates. The method is based on measuring the reflection coefficient of a composite consisting of a substrate, a patterned optical layer, the thin film to be monitored and the cover medium. The optical layer is patterned so that an array of squares is surrounded by the bare substrate. The reflectance difference between the optical layer squares and the bare substrate is the observable, whose fractional changes reveal the thickness of the film through a simple analytical expression. The periodic image is recorded by a digital microscope, and through Fourier transform techniques, the normalized differential reflectance of the patterned optical composite is calculated as the contrast factor of two dimensional bit map. The method is demonstrated by measuring a protein binding assay inside a microfluidic module placed under a microscope.

© 2013 OSA

## 1. Introduction

1. A. P. F. Turner, “Biochemistry. Biosensors--sense and sensitivity,” Science **290**(5495), 1315–1317 (2000). [CrossRef] [PubMed]

2. X. W. Guo, “Surface plasmon resonance based biosensor technique: a review,” J Biophotonics **5**(7), 483–501 (2012). [CrossRef] [PubMed]

3. S. Herranz, M. Bockova, M. D. Marazuela, J. Homola, and M. C. Moreno-Bondi, “Surface plasmon resonance biosensor for parallelized detection of protein biomarkers in diluted blood plasma,” Anal. Bioanal. Chem. **398**(6), 2625–2634 (2010). [CrossRef] [PubMed]

4. M. S. Luchansky and R. C. Bailey, “Silicon photonic microring resonators for quantitative cytokine detection and T-cell secretion analysis,” Anal. Chem. **82**(5), 1975–1981 (2010). [CrossRef] [PubMed]

5. A. Brecht, G. Gauglitz, and J. Polster, “Interferometric immunoassay in a FIA-system - a sensitive and rapid approach in label-free immunosensing,” Biosens. Bioelectron. **8**(7-8), 387–392 (1993). [CrossRef]

6. X. Zhu, J. P. Landry, Y. S. Sun, J. P. Gregg, K. S. Lam, and X. Guo, “Oblique-incidence reflectivity difference microscope for label-free high-throughput detection of biochemical reactions in a microarray format,” Appl. Opt. **46**(10), 1890–1895 (2007). [CrossRef] [PubMed]

7. J. Y. Wang, J. Dai, L. P. He, Y. Sun, H. B. Lu, K. J. Jin, and G. Z. Yang, “Label-free and real-time detections of the interactions of swine IgG with goat anti-swine IgG by oblique-incidence reflectivity difference technique,” J. Appl. Phys. **112**(6), 064702 (2012). [CrossRef]

## 2. Differential reflectance at normal incidence as an analytical tool

### 2.1. Optimization of the optical stack in terms of the reflectance sensitivity to film growth

_{0}, and substrate, n

_{3}), the film under study (n

_{1}, d

_{1}), and the optical layer (n

_{2}, d

_{2}) is shown in Fig. 1 .

_{i}(i = 1,2) are the normalized optical lengths in the film and optical layer, andwhere r

_{i}(i = 1,2,3) are the Fresnel coefficients, and

_{1}and use this parameter in normalized terms as an observable for the calculation of the film thickness. The selection of materials to optimize ∂R/∂d

_{1}is usually restricted to the optical layer (n

_{2}, d

_{2}) and the substrate (n

_{3}) since the cover medium is determined by the process to be followed for the film growth. For instance, if a protein binding assay is to be monitored, the cover medium will be the biological sample or assay buffer with n

_{0}≈1.34, while the protein layer has a refractive index n1 usually between 1.38 and 1.45. Therefore the emphasis from now on will be on how to maximize the magnitude of the derivative ∂R/∂d

_{1}in terms of (n

_{2}, d

_{2}) and n

_{3}. By differentiating Eq. (1) with respect to d

_{1}, we obtainWe will assume that δ

_{1}<< 1, that is, we are dealing with very thin films. Then Eq. (5) becomeswhereandOne first conclusion from Eq. (6) is that the sensitivity of the reflection coefficient to the monitored film goes to zero when r

_{1}= 0 (or n

_{0}= n

_{1}) since in this case there is no optical differentiation between the cover medium and the film under growth. More importantly, the same sensitivity vanishes when the optical layer (δ

_{2}, n

_{2}) is absent since in this case either δ

_{2}= 0 or r

_{3}= 0 andIn a patterned substrate, therefore, where the optical layer has been selectively removed, the bare substrate areas exhibit zero sensitivity to the film growth. On the contrary, in the areas bearing the optical layer the sensitivity of R to the grown film thickness is maximized if appropriate thickness δ

_{2}is chosen. To find the value of δ

_{2}(δ

_{2m}) that maximizes the absolute value of the derivative in Eq. (6) we set

_{2m})≈0, that is the optimum thickness is such that a photon that makes a round trip through the optical layer will experience a phase difference very close to π(1/2±m), with m an integer. Considering that |FR|«1 and |CR|«1 hold, or equivalently |r

_{1}|, |r

_{2}|«1, then the extrema for the derivative in Eq. (6) becomeIn Eq. (11) the sign of the derivative is minus when 2δ

_{2m}= (1/2 + 2m)π, or d

_{2}= (1/8 + 1/2m)λ

_{0}/n

_{2}, and plus when 2δ

_{2}= (3/2 + 2m)π, or d

_{2}= (3/8 + 1/2m)λ

_{0}/n

_{2}, with m an integer.

### 2.2. Differential reflectance as an intrinsic parameter in structured substrate characterization

_{1}to just measure the reflected beam on a one dimensional optical stack with optimized optical layer thickness. However, unwanted reflections in fluidic covers or various interfaces interfere with the measured sensitivity of R to d

_{1}. In fact they will underestimate of the actual value of the d

_{1}induced reflectivity changes if normalized with respect to the initial R value at d

_{1}= 0. In order to suppress unwanted reflection, we propose another method based on the measurement of the reflectance difference between sites with different optical layer thicknesses. In fact, the optical structure is partitioned into two interpolated array areas: one with no optical layer (bare substrate) where the initial reflectivity is R(0,0) and another bearing an optical layer with the optimized optical thickness δ

_{2m}=

_{2m}). If now the observable is the differential reflectance:the effects of parasitic and unwanted reflections are suppressed, especially when normalized to the initial value of ΔR. Differentiating Δ

*R*with respect to d

_{1}, and since the derivative of the first term of the right hand side in Eq. (12) is zero, we obtain with the help of Eq. (6)

_{2m}thickness is from Eq. (1)

_{2m})≈0 and that the absolute values of r

_{1}and r

_{2}are close to zero, an assumption usually valid for a realistic choice of materials, then

_{2}= (1/8 + 1/2m)λ

_{0}/n

_{2}, and minus when d

_{2}= (3/8 + 1/2m)λ

_{0}/n

_{2}. The accuracy of Eq. (16) is shown in Fig. 2 with δ

_{2}= δ

_{2m}= 3π/4 and it is compared to the exact expressions obtained by dividing the right hand sides of Eq. (13) and Eq. (14). Two different values and far apart were chosen for n

_{3}: 4 (silicon) and 1.46 (quartz). The value for the optical layer refractive index n

_{2}was swept from 1.4 to 2.2 covering the range of transparent dielectrics, while n

_{0}= 1.34 (bioassay buffer). As shown in Fig. 2, Eq. (16) is quite accurate and points out that the normalized differential reflectance is inversely proportional to the (n

_{0}-n

_{2}) difference and nearly independent of the substrate (n

_{3}). This is true especially for n

_{3}in the vicinity of n

_{2}, but even for n

_{3}= 4 it is still an excellent approximation for n

_{2}less than 1.5. Judging by the results from two different values of the film refractive index, n

_{1}= 1.4 and 1.45, we conclude that the sensitivity is proportional to the n

_{1}-n

_{0}difference (0.06 and 0.11 for the upper and the lower set of curves, respectively), as predicted by Eq. (16).

_{1}can be calculated dividing the fractional change of the measured differential reflectance by the sensitivity values in Fig. 2, provided of course that δ

_{1}«1 as is the case for biomolecular films of few nm. Using the simplified Eq. (16) we obtain:

_{0}, n

_{0}, n

_{1}, n

_{2}). Finally, the values of cos(2δ

_{2m}) from Eq. (10) are shown in Fig. 3 which proves that the approximation cos(2δ

_{2m})≈0 is very good for the range of values explored here.

### 2.3 Differential reflectance measurement through Fourier transform on periodic microscope images and choice of materials

_{0}= 1.34, n

_{2}= 1.46, n

_{3}= 4. For the protein refractive index two values were considered: n

_{1}= 1.4 and n

_{1}= 1.45. In a microscope a spectral band is more practically realized through the use of band pass optical filters as opposed to monochromatic light. The use of a narrow spectral band instead of monochromatic light has negligible effect on the differential reflectance sensitivity to film growth, as is shown in Fig. 4 . Here, the normalized differential reflectance sensitivity, calculated from Eq. (13) and Eq. (14) at λ

_{0}= 450 nm, is plotted along with the averaged differential reflectance sensitivity defined as

_{1}= 1nm and with ΔR calculated by averaging R(d

_{1},d

_{2m}), R(d

_{1},0) and R(0,d

_{2m}) from (1) over the spectral range 425-475nm. Therefore, the conclusions derived through the above equations obtained for monochromatic light still hold for narrow spectral bands where now λ

_{0}is the band center. Finally, for maximum sensitivity the oxide thickness is chosen as d

_{2m}= 3/8(λ

_{0}/n

_{2}) or δ

_{m}= 3π/4. With λ

_{0}= 450 nm, d

_{2m}assumes the value of 115 nm. Now, Eq. (16) holds with the minus sign which implies that protein binding decreases the differential reflectance.

## 3. Experimental method

### 3.1 Optical and bio-fluidic set up

_{2}layer was grown. The oxide layer was patterned through lithography and etching in buffered HF solution, so that a periodic pattern of oxide islands was created with a pitch of 20 microns (Fig. 5 ). The wafer was silanized with APTES and coated with biotinylated Bovine Serum Albumin (b-BSA). The total combined thickness of the optical layer (SiO

_{2}+ APTES + b-BSA) is estimated near 110 nm, very close to d

_{2m}= 115 nm.

### 3.2 Coating and Biomolecular reagents

9. M. Zavali, P. S. Petrou, S. E. Kakabakos, M. Kitsara, I. Raptis, K. Beltsios, and K. Misiakos, “Label-free kinetic study of biomolecular interactions by white light reflectance spectroscopy,” Micro & Nano Lett. **1**(2), 94–98 (2006). [CrossRef]

## 4. Experimental results and discussion

### 4.1. Contrast factor through Fourier transform

### 4.2 Bioanalytical results

_{1}= 1.4 or 4.6 nm in case n

_{1}= 1.45nm.

10. S. Busse, V. Scheumann, B. Menges, and S. Mittler, “Sensitivity studies for specific binding reactions using the biotin/streptavidin system by evanescent optical methods,” Biosens. Bioelectron. **17**(8), 704–710 (2002). [CrossRef] [PubMed]

11. P. Ihalainen and J. Peltonen, “Immobilization of streptavidin onto biotin-functionalized Langmuir-Schaefer binary monolayers chemisorbed on gold,” Sens. Actuat. B **102**(2), 207–218 (2004). [CrossRef]

^{−3}, 5.7x10

^{−4}and 2.8x10

^{−4}per shot, in good agreement with the concentration order.

### 4.3 Multianalyte capabilities

## 5. Conclusions

_{2}film. The only instrument employed here was a digital microscope plus an optical window equipped fluidic structure for the supply of reagents. After the introduction of the streptavidin solutions the signal changed proportionally to the analyte concentration and saturated at values in agreement to literature data for the same reaction. Multiplexed assay performance is possible if the illuminated field is partitioned into areas where different biomolecular probes have been immobilized and where independent resonant peaks are calculated.

## Acknowledgments

## References and links

1. | A. P. F. Turner, “Biochemistry. Biosensors--sense and sensitivity,” Science |

2. | X. W. Guo, “Surface plasmon resonance based biosensor technique: a review,” J Biophotonics |

3. | S. Herranz, M. Bockova, M. D. Marazuela, J. Homola, and M. C. Moreno-Bondi, “Surface plasmon resonance biosensor for parallelized detection of protein biomarkers in diluted blood plasma,” Anal. Bioanal. Chem. |

4. | M. S. Luchansky and R. C. Bailey, “Silicon photonic microring resonators for quantitative cytokine detection and T-cell secretion analysis,” Anal. Chem. |

5. | A. Brecht, G. Gauglitz, and J. Polster, “Interferometric immunoassay in a FIA-system - a sensitive and rapid approach in label-free immunosensing,” Biosens. Bioelectron. |

6. | X. Zhu, J. P. Landry, Y. S. Sun, J. P. Gregg, K. S. Lam, and X. Guo, “Oblique-incidence reflectivity difference microscope for label-free high-throughput detection of biochemical reactions in a microarray format,” Appl. Opt. |

7. | J. Y. Wang, J. Dai, L. P. He, Y. Sun, H. B. Lu, K. J. Jin, and G. Z. Yang, “Label-free and real-time detections of the interactions of swine IgG with goat anti-swine IgG by oblique-incidence reflectivity difference technique,” J. Appl. Phys. |

8. | O. S. Heavens, |

9. | M. Zavali, P. S. Petrou, S. E. Kakabakos, M. Kitsara, I. Raptis, K. Beltsios, and K. Misiakos, “Label-free kinetic study of biomolecular interactions by white light reflectance spectroscopy,” Micro & Nano Lett. |

10. | S. Busse, V. Scheumann, B. Menges, and S. Mittler, “Sensitivity studies for specific binding reactions using the biotin/streptavidin system by evanescent optical methods,” Biosens. Bioelectron. |

11. | P. Ihalainen and J. Peltonen, “Immobilization of streptavidin onto biotin-functionalized Langmuir-Schaefer binary monolayers chemisorbed on gold,” Sens. Actuat. B |

12. | M. Zavali, “Direct Study of biomolecular interactions through white light reflection spectroscopy” Senior Thesis, University of Ioannina, Greece, (2006). |

**OCIS Codes**

(100.0100) Image processing : Image processing

(100.2960) Image processing : Image analysis

(310.6860) Thin films : Thin films, optical properties

(280.4788) Remote sensing and sensors : Optical sensing and sensors

**ToC Category:**

Thin Films

**History**

Original Manuscript: October 23, 2012

Manuscript Accepted: January 9, 2013

Published: February 12, 2013

**Virtual Issues**

Vol. 8, Iss. 3 *Virtual Journal for Biomedical Optics*

**Citation**

I. Archontas, A. Salapatas, and K. Misiakos, "Molecular film growth monitoring via reflection microscopy on periodically patterned substrates," Opt. Express **21**, 4215-4227 (2013)

http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-21-4-4215

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### References

- A. P. F. Turner, “Biochemistry. Biosensors--sense and sensitivity,” Science290(5495), 1315–1317 (2000). [CrossRef] [PubMed]
- X. W. Guo, “Surface plasmon resonance based biosensor technique: a review,” J Biophotonics5(7), 483–501 (2012). [CrossRef] [PubMed]
- S. Herranz, M. Bockova, M. D. Marazuela, J. Homola, and M. C. Moreno-Bondi, “Surface plasmon resonance biosensor for parallelized detection of protein biomarkers in diluted blood plasma,” Anal. Bioanal. Chem.398(6), 2625–2634 (2010). [CrossRef] [PubMed]
- M. S. Luchansky and R. C. Bailey, “Silicon photonic microring resonators for quantitative cytokine detection and T-cell secretion analysis,” Anal. Chem.82(5), 1975–1981 (2010). [CrossRef] [PubMed]
- A. Brecht, G. Gauglitz, and J. Polster, “Interferometric immunoassay in a FIA-system - a sensitive and rapid approach in label-free immunosensing,” Biosens. Bioelectron.8(7-8), 387–392 (1993). [CrossRef]
- X. Zhu, J. P. Landry, Y. S. Sun, J. P. Gregg, K. S. Lam, and X. Guo, “Oblique-incidence reflectivity difference microscope for label-free high-throughput detection of biochemical reactions in a microarray format,” Appl. Opt.46(10), 1890–1895 (2007). [CrossRef] [PubMed]
- J. Y. Wang, J. Dai, L. P. He, Y. Sun, H. B. Lu, K. J. Jin, and G. Z. Yang, “Label-free and real-time detections of the interactions of swine IgG with goat anti-swine IgG by oblique-incidence reflectivity difference technique,” J. Appl. Phys.112(6), 064702 (2012). [CrossRef]
- O. S. Heavens, Optical Properties of Thin Solid Films (Dover Publications, 1991) Chap.4.
- M. Zavali, P. S. Petrou, S. E. Kakabakos, M. Kitsara, I. Raptis, K. Beltsios, and K. Misiakos, “Label-free kinetic study of biomolecular interactions by white light reflectance spectroscopy,” Micro & Nano Lett.1(2), 94–98 (2006). [CrossRef]
- S. Busse, V. Scheumann, B. Menges, and S. Mittler, “Sensitivity studies for specific binding reactions using the biotin/streptavidin system by evanescent optical methods,” Biosens. Bioelectron.17(8), 704–710 (2002). [CrossRef] [PubMed]
- P. Ihalainen and J. Peltonen, “Immobilization of streptavidin onto biotin-functionalized Langmuir-Schaefer binary monolayers chemisorbed on gold,” Sens. Actuat. B102(2), 207–218 (2004). [CrossRef]
- M. Zavali, “Direct Study of biomolecular interactions through white light reflection spectroscopy” Senior Thesis, University of Ioannina, Greece, (2006).

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