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In situ real-time monitoring of biomolecular interactions by using surface infrared spectroscopy
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Image of FIG. 1.
FIG. 1.

Biocells for MIR-IRAS measurements. The cells were used for monitoring (a) DNA and antigen-antibody interactions and (b) cultured living cells.

Image of FIG. 2.
FIG. 2.

IRAS spectra measured for (a) complementary ( and ) and (b) noncomplementary ( and ) pairs of ss-DNAs dissolved in containing 100 mM NaCl. (i) and (ii) are spectra for each ss-DNA. Thick lines of (iii) are spectra collected when the complementary and noncomplementary pairs of ss-DNAs were mixed. Thin lines of (iii) are calculated spectra obtained by summing up (i) and (ii). (c) Base pairs of DNA, adenine-thymine, and guanine-cytosine.

Image of FIG. 3.
FIG. 3.

IRAS spectra of the DNA-modified Si surfaces before (dotted lines) and after (solid lines) reaction with target DNA. Thick solid lines represent the corresponding difference spectra (solid line minus dotted line). The reference was the IRAS spectrum measured for the SSMCC-modified Si surface. Target DNA was (a) complementary ss-DNA and (b) noncomplementary ss-DNA . The thick solid lines are scaled up by a factor of 3.

Image of FIG. 4.
FIG. 4.

(a) Schematic of por-Si-based DNA microarray using IR microspectroscopy. [(b) and (c)] Typical scanning electron microscope (SEM) images of the por-Si-based microarray. (b) Top view of the array. (c) Cross-sectional view of the por-Si. (d) Immobilization of ss-DNA on the nanopore surfaces. Por-Si has a number of straight pores with a diameter of ca. 30 nm.

Image of FIG. 5.
FIG. 5.

(a) Top, IRAS spectrum of the -modified por-Si. Middle and bottom, IRAS spectra of -modified por-Si after reaction with (middle) or (bottom). The reference for the middle and bottom was the IRAS spectrum of the -modified por-Si. (b) IRAS spectra collected for , , and .

Image of FIG. 6.
FIG. 6.

Schematic of the working principle for a porous alumina-based DNA sensor. The illustrations are not to scale. (a) When a positive constant potential is applied to the Si prism, the DNA molecule moves into the vicinity of the prism surface and (b) when a negative constant potential is subsequently applied to the Si prism, the DNA is expelled from the Si prism surface.

Image of FIG. 7.
FIG. 7.

(a) Series of IRAS spectra for the complementary DNA pairs of and measured in containing NaCl at different solution temperatures. (b) Relationship between the solution temperature and the ratio of the IRAS peak intensity at to that at for the DNA pair of and . Denaturation curve for the DNA pair based on UV absorbance at 260 nm is also shown.

Image of FIG. 8.
FIG. 8.

(a) Hydrated with different bonding configurations of water molecules. (b) (i) Experimentally obtained IRAS spectrum of in in comparison with calculated vibration spectra (ii) of and (iii)–(vi) of hydrated with different configurations of molecules. (vii) was obtained by convoluting spectra (iii)–(vi).

Image of FIG. 9.
FIG. 9.

(a) MIR spectra in the midinfrared region for GaAs with an overlying layer. (b) Typical MIR absorption spectra of the antigen-immobilized (solid line) and antigen-unimmobilized (dotted line) sensors based on GaAs prisms with a layer. The spectra were measured in air. The reference was the spectra measured before the treatment of MPTMS.

Image of FIG. 10.
FIG. 10.

(a) Typical IR absorption spectra after addition of the antiserum with the antigen-immobilized (thick line) and antigen-unimmobilized (thin line) prisms. The references were the spectra before addition of the antiserum. The concentration of the antiserum was 1:1000 dilution. (b) Inverted second derivatives of (a) in the amide I region. [(c) and (d)] Antiserum concentration dependence on peak intensity at (c) 1639 and (d) . Values are mean ± SEM . Peak intensity 30 min after the addition of antiserum (open square) and that after washing out of the antiserum (solid square) is shown.

Image of FIG. 11.
FIG. 11.

Photomicrographs of (a) HL-60 cells incubated for 24 h and (b) MCF-7 cells incubated for 300 min in the sample chamber placed in the FTIR sample room. Scale bar: (a) 50 and (b) . (c) MIR-IRAS spectra of HL-60 cells obtained 5 h after (i) incubation with 0.2% Tween20 and (ii) incubation with cultured medium as a control experiment. (d) Time courses of peak intensity at (amide II band).


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752b84549af89a08dbdd7fdb8b9568b5 journal.articlezxybnytfddd
Scitation: In situ real-time monitoring of biomolecular interactions by using surface infrared spectroscopy