Elemental composition control of gold-titania nanocomposites by site-specific mineralization using artificial peptides and DNA

Biomineralization, the precipitation of various inorganic compounds in biological systems, can be regulated in terms of the size, morphology, and crystal structure of these compounds by biomolecules such as proteins and peptides. However, it is difficult to construct complex inorganic nanostructures because they precipitate randomly in solution. Here, we report that the elemental composition of inorganic nanocomposites can be controlled by site-specific mineralization by changing the number of two inorganic-precipitating peptides bound to DNA. With a focus on gold and titania, we constructed a gold-titania photocatalyst that responds to visible light excitation. Both microscale and macroscale observations revealed that the elemental composition of this gold-titania nanocomposite can be controlled in several ten nm by changing the DNA length and the number of peptide binding sites on the DNA. Furthermore, photocatalytic activity and cell death induction effect under visible light (>450 nm) irradiation of the manufactured gold-titania nanocomposite was higher than that of commercial gold-titania and titania. Thus, we have succeeded in forming titania precipitates on a DNA terminus and gold precipitates site-specifically on double-stranded DNA as intended. Such nanometer-scale control of biomineralization represent a powerful and efficient tool for use in nanotechnology, electronics, ecology, medical science, and biotechnology.


Dynamic light scattering (DLS) measurements
The sample solution (40 μL) was transferred into a UV-transparent disposable cuvette (S3, Sarstedt, Germany), and DLS data were acquired on a Zetasizer ZEN3600 instrument (Sysmex, Kobe, Japan) equipped with a 633-nm laser.

7-1. Titania precipitation sample
Samples incubated for 3 h were mixed with 9 µL of MilliQ water, and the entire volume of each sample was placed on a TEM grid (Cu 200 mesh covered with a collodion membrane, Nisshin EM, Tokyo, Japan) for 1 min and dried with filter paper. MilliQ water (20 µL) was then placed on the grid and immediately absorbed with filter paper. This process was repeated three times to remove salts from the sample. All samples were dried in vacuo prior to TEM measurements, which were conducted at an accelerating voltage of 200 kV (JEM-2100, JEOL, Tokyo, Japan). Stain samples were negatively stained with 2 % phosphotungstic acid.

7-2. Gold precipitation sample
Prior to gold precipitation, the gold precipitation sample solution (20 µL) was incubated for 12 h and then placed on a TEM grid for 1 min and dried with filter paper. MilliQ water (20 µL) was then placed on the grid and immediately absorbed with filter paper. This process was repeated three times to remove salts from the sample. All samples were dried in vacuo prior to TEM measurements, which were conducted at an accelerating voltage of 200 kV (JEM-2100, JEOL, Tokyo, Japan).

7-3. Gold-titania nanocomposite
The gold-titania sample (20 µL) was placed on a TEM grid for 1 min and dried with filter paper.
MilliQ water (20 µL) was then placed on the grid and immediately absorbed with filter paper. This process was repeated three times. All samples were dried in vacuo prior to TEM measurements, which were conducted at an accelerating voltage of 115 kV (JEM-1400, JEOL, Tokyo, Japan).
8. Gold-titania generated by conventional method 15 mL of 10 mM TiBALDH and 15 mL of 10 mM urea were added to 120 mL of MilliQ water, and incubated for 24 h at 160°C. Titania generated by centrifuging at 20,000 g for 30 min was recovered, and dried in vacuo [3] . 40 mL of 5 mM HAuCl 4 was mixed with 340 mL MilliQ water, and 20 mL of 20 mM NaBH 4 was added to 380 µL of HAuCl 4 solution. Incubated for 30 min at r.t. Gold nanoparticles generated by centrifuging at 20,000 g for 30 min were recovered, and dried in vacuo.
The dried titania and gold were placed in an aluminum crucible and calcined. During the calcination process, the temperature was increased from 25°C to 700°C within 20 min. The sample was calcined for 4 h in an air atmosphere and cooled to 37°C at a rate of 4.0°C min -1 .

Cell culture
Human cervical carcinoma (HeLa) cells were cultured in Dulbecco's modified Eagle's medium (D-MEM) supplemented with 10% fetal bovine serum and 1% penicillin streptomycin. HeLa cells were seeded with 2.5×10 4 cells in 24-well plate (Nest Biotechnology Co. Ltd., USA) and cultured at 37°C in a 5% CO 2 atmosphere for 24 h.

Cell viability measurements
HeLa cells were seeded with 2.5×10 4 cells in a 24-well plate and cultured at 37°C in a 5% CO 2 atmosphere for 24 h. 500 µL of 0.1 mg/mL titania sample in D-MEM were added to 24-well plates and incubated at 37°C in a 5% CO 2 atmosphere for 24 h. After incubation, the media were removed and the cells were washed three times with 1×PBS. The cells were then treated with 200 µL of 0.25% trypsin at 37°C in a 5% CO 2 atmosphere for 3 min. After adding 300 µL of the media, the cells were transferred into a microtube. The media were removed by centrifugation at 1,500 rpm for 5 min (room temperature). Cells were suspended in 50 µL of 1×HEPES, and transferred into a 96-well plate. 1×HEPES (40 µL) and Cell Counting Kit-8 (CCK-8, 10 µL, Dojindo Molecular Technologies, Tokyo, Japan) were added to the cell suspensions and kept at 37°C in a 5% CO 2 atmosphere for 1 h. The UV signal was monitored at 450 nm using a UV spectrophotometer (MTP-310 Microplate Reader, Colona Electric, Ibaraki, Japan).

Cell death induction under visible light irradiation
HeLa cells were seeded with 5.0×10 4 cells in a 24-well plate and cultured at 37°C in a 5% CO 2 atmosphere for 24 h. 500 µL of 0.1 mg/mL gold-titania samples in PBS were added to 24-well plates, and HeLa cells were irradiated with visible light (>450 nm) for 20 min. After incubation, the media were removed and the cells were washed three times with 1×PBS. The cells were then treated with 200 µL of 0.25% trypsin at 37°C in a 5% CO 2 atmosphere for 3 min. After adding 300 µL of the media, the cells were transferred into a microtube. The media were removed by centrifugation at 1,500 rpm for 5 min (room temperature). Cells were suspended in 50 µL of 1×HEPES, and transferred into a 96-well plate. 1×HEPES (40 µL) and CCK-8 (10 µL) were added to the cell suspensions and kept at 37°C in a 5% CO 2 atmosphere for 1 h. The UV signal was monitored at 450 nm using a UV spectrophotometer (MTP-310 Microplate Reader, Colona Electric, Ibaraki, Japan).

Absorption measurements
The sample solution (130 µL) was transferred into a quartz cuvette with a 1 cm pathlength. All absorption spectra data were acquired on a UV-1800 spectrometer (Shimadzu, Kyoto, Japan).

Atomic force microscopy (AFM)
The entire volume of each sample was placed on freshly cleaved mica (1 x 1 cm). After 5 min, the solvent was absorbed with filter paper. MilliQ water (20 µL) was then placed on the mica surface and immediately absorbed with filter paper. This process was repeated three times to remove salts from the sample. All samples were dried in vacuo prior to AFM measurements. Tapping-mode images were obtained on a MultiMode scanning probe microscope with a Nanoscope IIIa controller (Veeco, Woodbury, NY, USA).

Raman spectroscopy
Raman spectra were obtained with a confocal Raman spectrometer (RMP-510, JASCO, Tokyo, Japan). Mineralized titania by peptide was calcined with same protocol as that of the gold-titania nanocomposite and then the titania samples were placed on the stage of the spectrometer. The collection time was 10 s and the ranges of Raman shift were between 100 cm -1 and 1000 cm -1 .