Reaction mechanism of silane coupling on a SiO₂ surface is shown in Figure 1 [12]. Hypersil silica (GL Science Co., Ltd., Japan; particle diameter: 5 μm) was used as SiO₂ particles. Two kinds of silane derivatives were used; N-2-aminoethyl-3-aminopropyltrimethoxysilane (KBM-603; abbreviated to A-AM silane) and N-phenyl-3-aminopropyltrimethoxysilane (KBM-573; abbreviated to B-AM silane). Both chemicals were purchased from Shin-Etsu Chemical Co., Ltd., Japan. The structure was shown in Figure 2. Silanol derivatives were formed by hydrolysis of silane derivatives in aqueous solutions containing 2%, 20% and 40% A-AM or B-AM silane at 20 °C for 30 min. The silane coupling reaction of each silanol derivative was carried out at 100 °C for 10 min in a Teflon beaker. The hydroxyl groups of silanol derivatives were covalently bonded to hydroxyl groups on the SiO₂ surface via condensation reaction to form SiO₂ particles modified with aminoalkyl chains, [X-AM silane]/SiO₂ (X = A: amino group and B: phenyl group).

Functional groups on the surface of the aminoalkylated SiO₂ particles were qualitatively analyzed by Fourier transform infrared spectroscopy (FT-IR; Perkin Elmer, USA). Spectra were recorded using KBr discs containing 1 to 5 wt% sample. The substitution of aminoalkyl chains on the SiO₂ surface was further confirmed by fluorescence labeling. The reaction scheme is also illustrated in Figure 2. Each sample (5 mg) and fluorescein isothiocyanate (FITC; 2.5 μg/mL, Dojindo Laboratories Co., Ltd., Japan) in PBS solution was stirred at 20 °C for 5 min. The sample was washed twice with distilled water (DW) to give labeled aminoalkylated SiO₂ particles [FITC:X-AM silane]/SiO₂ (X = A: amino group and B: phenyl group). Fluorescence intensity was determined using a fluorescence microscope (U-RFLT50, Olympus, Japan) at a wavelength of 520 nm. [FITC:X-AM silane]/SiO₂ (5 mg) was dispersed in DW (300 μL) and placed in one well of a culture slide (well size: 10 mm², BD Co., Ltd., USA). After deposition of the particle solution for 5 min, images were obtained at 15 arbitrary points in the cell and analyzed to determine average intensity.

A flow chart outlining the preparation of complexes of single-stranded nucleic acid trapped with [A-AM silane]/SiO₂ and fluorescence compounds is shown in Figure 3. The schematic illustration was also shown in Figure 4. Single-stranded nucleic acids composed of only cytosine, adenine or guanine (poly-C, poly-A and poly-G, respectively; Wako Chemicals, Japan) were used. Solutions of poly-X (X = C, A or G, 12.5 mg/mL) in PBS were prepared. A mixture of each poly-X solution and [A-AM silane]/SiO₂ (5 mg) was incubated at 37 °C for 1 h. The particles were separated by centrifugation and then washed to give complexes of polynucleotide and [A-AM silane]/SiO₂ ([Poly-X:A-AM silane]/SiO₂; Figure 4a). An acridine orange derivative (named AO) was used as a fluorescent label (Cellstain-AO; Dojindo Laboratories Co., Ltd., Japan). The chemical structure is shown in Figure 4b. When AO interacts with phosphate group and/or nucleobase in single-stranded polynucleotides, such as complementary DNA or messenger RNA, the molecules are arranged in a random order. Some associate with AO in the single-stranded polynucleotide, giving rise to red fluorescence (λ_max = 650 nm) [13,14]. [Poly-X:A-AM silane]/SiO₂ (5 mg) was dispersed in AO in PBS solution (0.1–10 μg/mL) and incubated at 37 °C for 1 h. The particles were separated by centrifugation and washed to give fluorescent-labeled [Poly-X:A-AM silane]/SiO₂ particles ([AO:Poly-X:A-AM silane]/SiO₂; Figure 4c). Fluorescence intensity was measured using a fluorescence microscope at a wavelength of 640 nm using the method described in the previous section.

Molecular orbital (MO) calculations to obtain a surface model structure of aminoalkylated SiO₂, polynucleotides and fluorescent compound were carried out using commercial software (MOPAC Vr.3, Fujitsu Co., Japan) with the PM3 method [15]. A-AM silanol as a coupling precursor and a Si₄O₄(OH)₁₀ cluster combined with A-AM silanol as a surface model of a SiO₂ particle is shown in Figure 6a and b, respectively (silicon and oxygen atoms are shown as yellow and red color).

The geometry of each molecule was optimized, and then a hydroxyl group in each molecule was bonded to silica. The geometry was optimized again to generate an [A-AM silane]/SiO₂ cluster. Simulated FT-IR spectra were determined by vibration calculation of the [A-AM silane]/SiO₂ cluster using the PM3 method. Polynucleotide composed of three units of nucleobase and cellstain-AO molecule as a fluorescent compound was also calculated by the method described above.

FT-IR spectra of the surface of SiO₂ particles after aminosilane coupling are presented in Figure 5. Here, SiO₂ particles treated with X-AM silane (n%) are abbreviated as [X-AM silane (n%)]/SiO₂. Figure 5a shows an FT-IR spectrum of [A-AM silane (2%)]/SiO₂. The peaks labeled 1 and 5 are assigned to -OH (ν = 3385–3418 cm⁻¹) and Si-O-Si bonds (ν = 1090–1120 cm⁻¹) in SiO₂. Peaks 2 and 3 are assigned to -CH₂- (ν = 2911–2929 cm⁻¹) and -NH₂ or -CH₂-NH₂ bonds (ν = 1568–1634 cm⁻¹), respectively. Figure 5b shows an FT-IR spectrum of [A-AM silane (40%)]/SiO₂, which contains Peak 4 assigned to -CH₂- bonds (ν = 1471 cm⁻¹). These results suggest that A-AM silane has been introduced onto the surface of SiO₂. Figure 5c shows an FT-IR spectrum of [B-AM silane (2%)]/SiO₂ for comparison. In this case, peak 3 almost disappeared because there is no amino group in B-AM silane.

Measured and calculated FT-IR spectra are presented in Figure 6, where a spectrum measured for [A-AM silane (40%)]/SiO₂ (Figure 6c) is compared to simulated spectra for A-AM silanol (Figure 6a) and [A-AM silane]/SiO₂ clusters (Figure 6b).

Six major peaks were obtained from the vibrational calculation for A-AM silanol, as shown in Figure 6a. Peaks 2, 4, 6 and 7 are assigned to -CH₂- bonds (ν = 3022, 1376, 1160 and 779 cm⁻¹), while peaks 1 and 3 are assigned to -NH₂ bonds (ν = 3,522 and 1,670 cm⁻¹). Except for peak 5, these correspond to the calculated spectrum for an [A-AM silane]/SiO₂ cluster (Figure 6b). Peak 5 for the cluster is consistent with vibration of Si-O-Si bonds (ν = 965 cm⁻¹). The wavenumbers of the simulated peaks are close to those in an FT-IR spectrum measured for [A-AM silane (40%)]/SiO₂ (Figure 6c).

Figure 7 shows the relationship between the results of the vibrational calculation for [A-AM silane]/SiO₂ cluster as a model molecule (Figure 6b) and the measured data for [A-AM silane (40%)]/SiO₂ (Figure 6c). There is a good correlation between the simulated and measured peaks. Fitting the data with a linear approximation gave a correlation coefficient of 0.9948. This suggests that the structure of the aminoalkyl chains on the SiO₂ surface is similar to the structure predicted for the [A-AM silane]/SiO₂ cluster.

Figure 8 shows fluorescence microscope images of [B-AM silane (2%)]/SiO₂ (Figure 8a) and [A-AM silane (2%)]/SiO₂ (Figure 8b) after reaction with FITC, and models of the surface structure of both particles are shown in Figure 8c and d, respectively. The isothiocyanate group in FITC reacts selectively with a primary amine group, such as that in A-AM silane to form a thiourea bond between both functional groups.

The intensity of green fluorescence at λ = 520 nm from the samples is clearly different. [A-AM silane (2%)]/SiO₂ treated with FITC shows a relative fluorescence intensity of 250 ± 4, whereas for [B-AM silane (2%)]/SiO₂ it is 50 ± 4. These results suggest that the terminally-connected phenyl group in B-AM silane and amino group in A-AM silane are oriented outwards on the surface of the SiO₂ particles.

Figure 9 shows the relationship between the concentration of AO and fluorescence intensity of Poly-X (X = guanine (G), adenine (A) and cytosine (C)) conjugated with [A-AM silane]/SiO₂. The schematic illustrations are also shown in Figure 4c. The fluorescence intensity of [A-AM silane]/SiO₂ treated with AO was measured to confirm their interaction. There was no interaction between AO and [A-AM silane]/SiO₂ (dotted line in Figure 9). The intensity of the red fluorescence at λ = 640 nm clearly depended on the type of polynucleotide in the conjugate. When different concentrations of AO were conjugated with [Poly-G:A-AM silane]/SiO₂, the relative fluorescence intensity of [AO:Poly-G:A-AM silane]/SiO₂ was 45 for 1 μg/mL, 129 for 5 μg/mL and 177 for 10 μg/mL. When AO was conjugated with [Poly-A: A-AM silane]/SiO₂, the fluorescence intensity of [AO: Poly-A: A-AM silane]/SiO₂ decreased compared with that of [AO: Poly-G: A-AM silane]/SiO₂ to 41 for 1 μg/mL, 81 for 5 μg/mL and 144 for 10 μg/mL. In the case of Poly-C, the fluorescence intensity of [AO:Poly-C:A-AM silane]/SiO₂ increased very little with concentration, showing an intensity of 27 for 1 μg/mL, 43 for 5 μg/mL and 54 for 10 μg/mL.

The sensitivities of fluorescence intensity, R, of AO-labeled [Poly-X: A-AM silane]/SiO₂ conjugates are summarized in Table 1. For comparison, R of AO-labeled Poly-X ([AO: Poly-X]) are also summarized in Table 1. R was calculated using Equation (1):

(1)

In this instance, AO₁₀ and AO₁ were the concentrations of AO (μg/mL), while FL₁₀ and FL₁ were the fluorescence intensities when the concentrations of AO were 10 and 1 μg/mL, respectively. The difference of conjugation ability is particularly clear when single-stranded nucleic acids trapped with aminoalkylated SiO₂ are used. R of [Poly-X:A-AM silane]/SiO₂ increased in the order Poly-C < Poly-A < Poly-G. A similar trend was obtained for the [AO:Poly-X] complexes (amino-alkylated SiO₂ particle free). These results suggest that the conjugation of AO and [Poly-X:A-AM silane]/SiO₂ is affected by the characteristics of the single-stranded nucleic acid. Based on the above results, it estimates that the difference of fluorescence intensity for AO and [Poly-X:A-AM silane]/SiO₂ was obtained clearly when the amount of each poly-X on amino-alkylated SiO₂ particle was the same.

Electron distributions of each model structure, which a surface model of a [A-AM silane]/SiO₂ cluster (Figure 6b), Poly-G, A or C with 3 units as the nucleotide fragment (Table 2) and fluorescence compound (Figure 4b), were calculated separately by the PM3 method.

Based on the results of electron distributions, the highest occupied molecular orbital (HOMO) is distributed on the terminal of each [A-AM silane]/SiO₂ cluster. This is because the nitrogen atom in an amino group has a lone electron pair. This result suggests that the surface of aminoalkylated SiO₂ has an electron-donating ability. In PBS solution (pH = 7), protonation of the primary amino group is an unfavorable reaction. It estimates there is little positive electric charge on the surface of the silica particle.

In contrast, it has been reported that AO as a fluorescence compound interacts with the phosphate group on the nucleotide by electrostatic interaction [13,14]. In accordance with the MO calculation result, the HOMO of AO, which contains a π electron, is also delocalized over the entire molecule. This suggests that AO also possesses electron-donating ability. It estimates that AO attaches to a phosphate group in the nucleotide by electrostatic interaction first, then the nucleobase around the AO interacts through its electron-donating ability.

Based on the results in Figure 9 and Table 1, the polynucleotides conjugate was trapped with aminoalkylated SiO₂ and was further conjugated with AO. This suggests that the polynucleotide behaves as an electron acceptor, and two vacant orbitals are necessary to interact with both aminoalkylated SiO₂ and AO. Based on the results of electron distributions, the lowest unoccupied molecular orbital (LUMO) and second LUMO of the nucleotide fragment are distributed on a central nucleotide and 5’ end.

The interaction between the nucleotide and AO was evaluated using Equation (2):

(2)

In this instance, E_N is the energy level of the LUMO obtained from a MO calculation of each nucleotide, E_A is energy level of the HOMO obtained from the MO calculation of AO, and ΔE is an energy gap between each nucleotide conjugated and AO. Therefore, it is possible to determine an interaction parameter between two molecules using Frontier orbital theory [16].

A comparison of ΔE values is given in Table 2. ΔE decreased in the order of Poly-C > Poly-A > Poly-G, consistent with the results presented in Figure 9. This order reflects the interaction between fluorescent compounds and the nucleobase in single-stranded nucleic acids.