LABEL-FREE DETECTION OF DNA USING DIFFRACTED LASER IN NANOWALL ARRAY STRUCTURES

Takao Yasui1,2*, Noritada Kaji3, Yukihiro Okamoto2, Manabu Tokeshi1,2, Yasuhiro Horiike4 and Yoshinobu Baba1,2,5

1Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, JAPAN, 2FIRST Research Center for Innovative Nanobiodevices, Nagoya University, JAPAN,

3Graduate School of Science, ERATO Higashiyama Live-Holonics Project, Nagoya University, JAPAN, 4National Institute for Materials Science, JAPAN, and

5Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), JAPAN ABSTRACT

We developed a new label-free system using nanowall array structures and realized label-free detection of small molecules and DNA. For the achievement of label-free detection, we used the diffracted laser beam, which was produced by the nanowall array structures when the laser was passed through the nanowall array structures perpendicularly. The diffracted signals showed a good linear relationship between the signal intensity and refractive indices, concentrations of molecules, or DNA length.

KEYWORDS: Nanowall, Label-Free Detection, Diffracted Laser

INTRODUCTION

Recently, many researchers have paid attention to micro-total-analysis-systems (µTAS) devices due to an advantage which these devices could integrate whole sets of laboratory procedures in biotechnology and chemical analysis. Especially, during the past decades, numerous nanostructures to separate DNA or protein molecules have been reported [1], since they have tremendous advantages to realize ultra-fast analysis of biomolecules with ultra small sample consumption, compared with the conventional gel-based separation technologies such as agarose or polyacrylamide gel electrophoresis. While these nanostructures made a great contribution to the separation science, many researchers have been forced to be required an extra preparation step to label the samples with fluorophores. Carrying out the seamless analysis from sample preparation to detection in a chip, it is preferable that we do not need any extra preparation steps for labeling, i.e., the technique of label-free detection without fluorescent tags has been desired. And also, the label-free detection might provide another benefit to the clinical application of biomolecular samples. If we could attain the label-free detection of rare target analyte, which separated and extracted from samples, and identify them, we would apply them to the clinical application.

In this paper, we demonstrated a label-free detection method using nanostructures without any fluorescent molecules. This method we developed uses the light-diffraction phenomenon which nanostructures have intrinsically. Another benefit using nanostructures is that they have a potential to be a sieving matrix for biomolecules, simultaneously.

THEORY

Our detection system is based on the diffraction of the incident laser. Briefly, the experimental setup mainly consists of a laser, a ND filter, a light chopper, an objective lens, a nanowall array chip, a photodiode, and a lock-in amplifier, as shown in elsewhere [2]. The nanowall array structures, which is 600 nm width, 2.7 µm height and 200 nm spacing between nanowalls, was regarded as a diffraction grating. In above structures, the grating pitch was thought to be 800 nm, the incident laser of 532 nm seemed to be diffracted in nanowall array strucutures. The diffraction condition is expressed below;

!

psin" = n# (1)

where p is the pitch of the grating (800 nm), θ is the diffractive angle, n is an integer number, and λ is the wavelength of the incident laser (532 nm). From the above equation, the diffracted angle of 27º was calculated at the first order of diffraction.

In this time, we used 10×/0.30NA objective lens to focus the incident laser of 532 nm. Under these conditions, the radius of laser spot, r, was calculated by using equation (2);

!

r =0.61" #NA

(2)

where NA is the numerical aperture. The equation (2) showed the radius of laser spot of 1081 nm. In the diameter of

2162 nm, there are two or three nanowalls and then the active area that samples can pass through has a range of 8.02×105 ≤ S

978-0-9798064-4-5/µTAS 2011/$20©11CBMS-0001 54 15th International Conference onMiniaturized Systems for Chemistry and Life Sciences

October 2-6, 2011, Seattle, Washington, USA

≤ 1.01×106 (nm2). Because the nanowall array has the height of 2.7 µm, the valid volume of detection has a range of 2.17×109 ≤ V ≤ 2.73×109 (nm3), around 2 fL.

EXPERIMENTAL

In this study, a nanowall array, which is 600 nm width and 2.7 µm height, was fabricated on a quartz substrate by electron beam lithography, photolithography, and plasma etching, as described elsewhere [3]. We precisely controlled the spacing of 200 nm between nanowalls. We used 532 nm emission line of a diode pumped solid-state laser with output power of 20 mW for the incident laser. A light chopper modulated the incident laser with a modulation frequency at 1013 Hz. The laser was focused by a 10×/0.30NA objective lens. The focused laser was diffracted by nanowall array structures at the diffraction angle of 27º in the first diffraction. The diffracted laser was detected by a photodiode and fed into a lock-in amplifier. The time constant of the lock-in amplifier was set to 100 ms. To get the effective signal change, we set the photodiode to get the local maximum signal.

For the performance assessment of our label-free detection system, we used double distilled water (ddH2O), methanol, acetone, tetrahydrofuran, and isopropanol. At 589 nm, the refractive index of air, methanol, ddH2O, acetone, tetrahydrofran, and isopropanol is 1.0002, 1.3284, 1.3330, 1.3586, 1.3772, and 1.4072, respectively [4]. For the absorbance experiments, we used two types of absorbance molecules; rhodamine 6G (R6G) and sunset yellow FCF (SY). R6G transferred the absorbed energy to fluorescence or heat (thermal energy), and SY did it to only heat. The absorption spectra of R6G and SY showed that R6G has a local maximum at 532 nm but SY does not. DNA molecules also were used for the non-absorbance molecules. To avoid any conformational changes of DNA molecules, we used DNA molecules below 500 bp, which contour length was 170 nm.

RESULTS AND DISCUSSION

Verification of an detection ability of our label-free system was our first approach. To examine it, we used the detection of the signal change from air (1.0002) to water (1.3330). After drying out, we set the nanowall array chip to get the label-free signal in air condition, and then we gradually filled ddH2O by using capillary forces. As shown in Figure 1(a), the signal intensity of label-free detection decreased as the ddH2O filled inside nanowall array structures, and finally the signal intensity reached a plateau in the condition that all nanowall array regions were filled with ddH2O.

To reveal the contribution of the difference of refractive indices to label-free detection, we quantitatively investigated the signal intensity from air (= 1.0002) to each solvent in Figure 1(b). As the difference of refractive indices between air and solvent increased, the signal variances increased in response to refractive indices and showed a good linear relationship.

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Figure 1: (a) The signal change of label-free detection from air to ddH2O. The signal intensity rapidly decreased as ddH2O was introduced to nanowall array structures by capillary forces. (b) The dependence of refractive indices on signal variances of label-free signals. Red dotted lines show linear approximations (N=5).

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Figure 2: (a) Signal intensity of label-free detection vs. concentration of sunset yellow FCF molecules (n = 3). Dotted lines show a linear approximation. (b) The dependence of the length (bp) of DNA molecules on signal intensity. The concentration of each DNA molecule is the same value (1.52 µM). The dotted line shows a linear approximation (N=5).

Molecular absorption is also a conceivable factor to affect label-free detection. Next, we monitored the label-free signal

change of SY in the concentration range from 10 to 1000 µM and R6G from 10 to 100 µM (data not shown). The plots of SY (Figure 2(a)) showed a linear relationship between signal variance and concentrations. In Figure 2(b), we showed the results of label-free detection of DNA molecules. A linear relationship between signal intensity and DNA length obviously exhibited. The difference of refractive index or absorption of DNA molecules might make a contribution to these signal intensities.

CONCLUSION

Consequently, we set up a new method for label-free detection using the diffracted laser derived from the nanowall array structures. Our label-free system could detect the variance from air to water, or solvents, absorbance molecules (SY), and DNA molecules. In the future, owing to the availability to separate DNA molecules in the nanowall chips, it is feasible to separate and detect label-free DNA molecules simultaneously with the nanowall chips. ACKNOWLEDGEMENTS

This research is partially supported by the Japan Society for the Promotion of Science (JSPS) through its “Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program)" and Grant-in-Aid for JSPS Fellows. We also thank Dr. Ryo Ogawa and Dr. Shingi Hashioka for their technical supports. REFERENCES [1] N. Kaji, Y. Okamoto, M. Tokeshi, and Y. Baba, “Nanopillar, Nanoball, and Nanofibers for Highly Efficient Analysis of

Biomolecules,” Chem. Soc. Rev., vol. 39, pp 948-956, 2010.; G.B. Salieb-Beugelaar, K.D. Dorfman, A. van den Berg, J.C. Eijkel, “Electrophoretic Separation of DNA in Gels and Nanostructures,” Lab Chip, vol. 9, pp. 2508-2523, 2009.

[2] T. Yasui, N. Kaji, Y. Okamoto, M. Tokeshi, Y. Horiike, and Y. Baba, “Label-Free Detection of Biomolecules with Nanowall Arrays,” Proc. Micro Total Analysis Systems 2010, pp. 485-487, 2010

[3] N. Kaji, Y. Tezuka, Y. Takamura, M. Ueda, T. Nishimoto, H. Nakanishi, Y. Horiike, and Y. Baba, “Separation of Long DNA Molecules by Quartz Nanopillar Chips under a Direct Current Electric Field,” Anal. Chem., vol. 76, pp 15-22, 2004.; T. Yasui, N. Kaji, R. Ogawa, S. Hashioka, M. Tokeshi, Y. Horiike, and Y. Baba, “DNA Separation by Square Patterned Nanopillar Chips,” Proc. Micro Total Analysis Systems 2007, vol. 2, pp. 1207-1209, 2007.

[4] J. Rheims, J. Kӧser, and T. Weiedt, “Refractive-Index Measurements in the Near-IR Using an Abbe Refractometer,” Meas. Sci. Technol., vol. 8, pp. 601-605, 1997.

CONTACT *Takao Yasui, tel: +81-52-7893560; yasui.takao@e.mbox.nagoya-u.ac.jp

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