Label-free detection of real-time DNA amplification using a nanofluidic diffraction grating

Abstract

Quantitative DNA amplification using fluorescence labeling has played an important role in the recent, rapid progress of basic medical and molecular biological research. Here we report a label-free detection of real-time DNA amplification using a nanofluidic diffraction grating. Our detection system observed intensity changes during DNA amplification of diffracted light derived from the passage of a laser beam through nanochannels embedded in a microchannel. Numerical simulations revealed that the diffracted light intensity change in the nanofluidic diffraction grating was attributed to the change of refractive index. We showed the first case reported to date for label-free detection of real-time DNA amplification, such as specific DNA sequences from tubercle bacilli (TB) and human papillomavirus (HPV). Since our developed system allows quantification of the initial concentration of amplified DNA molecules ranging from 1 fM to 1 pM, we expect that it will offer a new strategy for developing fundamental techniques of medical applications.

Figure 1. Label-free detection system using the nanofluidic diffraction grating.

(a) A photo of the nanofluidic diffraction grating; scale bar, 1 cm. Interference fringes indicated the nanochannels were embedded in the microchannel as marked by the white arrow. (b) A SEM image of the nanochannels, which was 800 nm period, and 200 nm width and 2.7 μm depth fused silica nanogrooves; scale bar, 1 μm. (c) A schematic illustration showing the setup for label-free detection using signal changes of the diffracted light when the nanofluidic diffraction grating was filled with a sample (various liquids, DNA molecules, or amplified DNA molecules). (d) A photo showing the diffracted light (white arrows) and the origin of the beam; scale bar, 1 cm. The white dotted lines outline the nanofluidic diffraction grating. (e) A two-dimensional (2D) intensity profile of the diffracted light; scale bar, 1 mm. Color gradation showing intensity variation; red and black colors mean high and low intensities, respectively. (f) A schematic illustration showing the diffraction of light through the device. (g) Transmissivity for the wavelength of 532 nm at each output angle. The 532 nm laser beam has some transmittance at 42°.

Figure 2. Label-free detection of various liquids introduced into the nanofluidic diffraction grating.

(a) A schematic illustration showing introduction of various liquids into the nanochannels by capillary force. (b) Time-course monitoring of normalized ∆I of the diffracted light during water introduction. Normalized ∆I was defined as the intensity difference from the initial state after sample introduction. (c) A micrograph of the nanochannels as indicated by the double-headed arrow before water introduction; scale bar, 50 μm. (d) A micrograph of the nanochannels as indicated by the double-headed arrow after water introduction; scale bar, 50 μm. (e) Intensity profile of the diffracted light before water introduction; scale bar, 1 mm. (f) Intensity profile of the diffracted light after water introduction; scale bar, 1 mm. (g) Normalized ∆I plot derived from signal changes by the transit of various liquids, such as methanol, water, acetone, ethanol, isopropanol, tetrahydrofuran, cyclohexane, chloroform, toluene, o-xylene, and chlorobenzene, through the nanochannels, with respect to the refractive indices of them. Error bars show the standard deviation for a series of measurements (N = 5). (h) Diffraction efficiency derived from RCWA versus the refractive indices of the various liquids. The diffraction efficiency calculated by the RCWA simulation showed the percentage for an allocated intensity of the diffracted light of the first order based on the intensity of the incident laser beam.

Figure 3. Label-free detection of real-time DNA amplification in the nanofluidic diffraction grating.

(a) A schematic illustration showing real-time DNA amplification in the device at 34 °C. (b) Time-course monitoring of normalized ∆I of the diffracted light during real-time DNA amplification with different target sequences. Target sequences of amplified DNA molecules were human papillomavirus (HPV, red circles, initial concentration: 1 pM) sequence and tubercle bacilli (TB, blue circles, initial concentration: 1 pM) sequence. Negative control data (black circles) were obtained with no target sequence. Error bars show the standard deviation for a series of measurements (N = 3). (c) Refractive index versus amplification time. The initial concentration of TB sequence was 1 pM. (d) Diffraction efficiency derived from RCWA versus amplification time. The initial concentration of TB sequence was 1 pM. (e) Time-course monitoring of normalized ∆I of the diffracted light during real-time DNA amplification for TB sequence with different initial concentrations. Negative control data (black circles) were obtained with no target sequence. Error bars show the standard deviation for a series of measurements (N = 3). (f) Normalized ∆I plot derived from signal changes by DNA amplification for 15 min in the nanochannels, with respect to the initial concentration of TB sequence. Error bars show the standard deviation for a series of measurements (N = 3).