Plasmonic Manipulation of DNA using a Combination of Optical and Thermophoretic Forces: Separation of Different-Sized DNA from Mixture Solution

We demonstrate the size-dependent separation and permanent immobilization of DNA on plasmonic substrates by means of plasmonic optical tweezers. We found that a gold nanopyramidal dimer array enhanced the optical force exerted on the DNA, leading to permanent immobilization of the DNA on the plasmonic substrate. The immobilization was realized by a combination of the plasmon-enhanced optical force and the thermophoretic force induced by a photothermal effect of the plasmons. In this study, we applied this phenomenon to the separation and fixation of size-different DNA. During plasmon excitation, DNA strands of different sizes became permanently immobilized on the plasmonic substrate forming micro-rings of DNA. The diameter of the ring was larger for longer DNA (in base pairs). When we used plasmonic optical tweezers to trap DNA of two different lengths dissolved in solution (φx DNA (5.4 kbp) and λ-DNA (48.5 kbp), or φx DNA and T4 DNA (166 kbp)), the DNA were immobilized, creating a double micro-ring pattern. The DNA were optically separated and immobilized in the double ring, with the shorter sized DNA and the larger one forming the smaller and larger rings, respectively. This phenomenon can be quantitatively explained as being due to a combination of the plasmon-enhanced optical force and the thermophoretic force. Our plasmonic optical tweezers open up a new avenue for the separation and immobilization of DNA, foreshadowing the emergence of optical separation and fixation of biomolecules such as proteins and other ncuelic acids.


Plasmonic nanostructures
We fabricated a a gold nanopyramidal dimer array on a glass substrate by means of angular-resolved nanosphere lithography (AR-NSL). [1][2][3] Representative polarized extinction spectra shows Fig. S1(a). The plasmonic substrate has a broad extinction band around 800 nm, which is ascribed to a gap-mode plasmonic resonance of gold nanopyramids ( Fig. S1(b) 4 ). We have already discussed the enhancement effect of the plasmonic nanostructures by means of theoretical calculation (discrete dipole approximation) in elsewhere [4][5][6] . The electric field of resonant light (λ = 808 nm) is strongly enhanced at the nanogaps between the nanopyramids.

Fig. S1
(a) Representative polarized extinction spectra from an AR-NSL substrate. The polarized angles of incident light were parallel (blue) and perpendicular (red) to the long axis of the dimers, respectively.

Optical setup for plasmonic optical tweezers combined with confocal fluorescence microspectrosocpy
We have already described the detail of optical setup for plasmonic optical tweezers combined with confocal fluorescence microspectroscopy in elsewhere 5,7,8 Fig. S3 shows bright-field micrographs during plasmonic optical trapping of λ-DNA by changing plasmon excitation intensity. We successfully formed a DNA micro-ring on a plasmon substrate in a intensity range of 10 -20 kW/cm 2 . Below 10 kW/cm 2 , we have never observed any sign during plasmon excitation. Over 20 kW/cm 2 , a micro-bubble formation was observed at the irradiation area by a local photothermal effect of the excited gold nanostructures.

DNA micro-ring formation by changing excitation intensity and irradiation area
The diameters of the micro-rings (D ring ) were evaluated to be 7.5 µm at 13 kW/cm 2 , 8.3 µm at 15 kW/cm 2 , 9.1 µm at 17 kW/cm 2 , and 9.8 µm at 20 kW/cm 2 . These results indicated that the micro-ring size was controllable by changing plasmon excitation intensity. The micro-ring size was also controllable by changing the size of irradiation area. Fig.   S4 shows bright-field micrographs during plasmonic optical trapping of λ-DNA by changing the size of irradiation area. D ring of the micro-ring became larger with increasing the irradiation area: (a) 4.6 µm (26 µm 2 ), 6.5 µm (35 µm 2 ), 7.9 µm (45 µm 2 ), and 8.3 mm (51 µm 2 ).

Fig. S4
Bright-field micrographs of plasmonic optical trapping of λ-DNA by changing irradiation area:

Experimental determination of temperature elevation during plasmon excitation
We determined temperature elevation to obtain fluorescence spectra of a thermoresponsive fluorescence dye (2',7'-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein, BCECF) located on a plasmonic substrate during plasmon excitation. BCECF has fluorescence intensity sensitive to temperature. The fluorescence intensity of the dye decreased by 7.8 % when temperature increased by 10 K. Using this molecular probe, we precisely determined temperature elevation during plasmon excitation for obtaining temperature gradient around the plasmon excitation area. Fig. S6(a) shows fluorescence spectra of BCECF at the center of plasmon excitation area. Increasing plasmon excitation intensity, the fluorescence intensity decreased. We determined temperature elevation ∆T from the room temperature ( Fig. S6(b)) based on the fluorescence spectra ( Fig. S6(a)). At 15 kW/cm 2 , we determined ∆T to be 41 K. By using this technique for temperature gradient around the plasmon excitation area, we also obtained the spatially resolved fluorescence spectra during plasmon excitation at each distance r from the center of the area to the outer-side of the area (Fig. S6(c)). Fig. S6(d) shows temperature elevation ∆T at distance r by changing plasmon excitation intensity. The slopes of these fitting curves correspond to the spatial gradient of temperature. We determined a spatial temperature gradient to be ∇T= − 3.4 K/µm at 15 kW/cm 2 .