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Photoluminescence behaviors of morin–human immunoglobulin on porous anodized aluminum oxide films
Thin Solid Films 471 (2005) 264 – 269 www.elsevier.com/locate/tsf
Photoluminescence behaviors of morin–human immunoglobulin on porous anodized aluminum oxide films R.P. Jia a, Y. Shen a, H.Q. Luo a, X.G. Chen a,*, Z.D. Hu a, D.S. Xue b a
b
Department of Chemistry, Lanzhou University, Lanzhou 730000, PR China Key Lab for Magnetism and Magnetic Materials of MOE, Lanzhou University, Lanzhou 730000, PR China Received 8 October 2003; received in revised form 25 May 2004; accepted 9 June 2004 Available online 19 July 2004
Abstract For the first time, the photoluminescence (PL) spectra of porous anodized aluminum oxide (AAO) films impregnated with essentially non-fluorescent 3, 5, 7, 2V, 4V-pentahydroxyflavone (morin) and morin – human immunoglobulin (IgG) were investigated and compared with those of liquid solutions. It was found that their PL band positions are similar to that of morin – Al3 + in ethanol solution, and the PL intensity of embedded morin alone can be enhanced greatly by the introduction of human immunoglobulin. We infer that the appearance of the PL band detected here is due to the formation of morin – Al complex in the holes of AAO with the inner wall involved, and a likely luminescent mechanism is proposed to elucidate the PL enhancement phenomena due to the coexistence of morin and IgG in the AAO pores, which is confirmed by ultraviolet (UV) – visible and Fourier transform infrared (FTIR) measurements. Moreover, it is also found that the PL intensity increases with the pore size. D 2004 Elsevier B.V. All rights reserved. Keywords: Photoluminescence; Morin; IgG; AAO film
1. Introduction Porous anodized aluminum oxide (AAO), a typical selfordered nano-channel material formed by anodizing Al in an appropriate acid solution, has recently stimulated considerable interest as a key material for the fabrication of nanometer-scale structures [1 – 3]. The structure of AAO is described as a closely packed array of columnar cells, each containing a central pore of which the size and interval can be controlled by changing the forming conditions [4]. In recent years, a few attempts have been focused on the optical properties of luminescent substances embedded in the pores of AAO layer, which have shown promising applications in novel optoelectronic devices, such as microcavities, solid state lasers and so on [5,6]. However, as far as we know, there is no report on the luminescent properties and corresponding luminescent mechanisms of small organ-
* Corresponding author. Fax: +86-9318912582. E-mail addresses: [email protected], [email protected] (X.G. Chen). 0040-6090/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.tsf.2004.06.113
ic dye molecule and bio-macromolecule impregnated in the pores of AAO films. 3, 5, 7, 2V, 4V-pentahydroxyflavone (morin, its structure is shown in Fig. 1), a kind of O, O-donating chelating reagent, is only weakly fluorescent by itself but forms highly fluorescent complex with Al3 +. Based on this, a sensitive method for the determination of aluminum was developed; the detection limit can be reached to the parts-per-billion range [7,8]. Moreover, this dye also has been used as a probe for other metal ions [9,10] and some bio-macromolecule, such as protein [11] and nucleic acid [12] by the liquid phase. The aim of this work is to draw attention to a complementary approach that utilizes AAO technology to develop the luminescence of small organic molecules and bio-macromolecules in a nanometer-sized structure. Based on the above-mentioned remarks, in this paper, we investigated for the first time the photoluminescence (PL) properties of AAO films immersed in solutions containing dye and human immunoglobulin (IgG) or not, and a possible luminescence mechanism is proposed to explain the PL enhancement. We hope that the proposed approach would be useful to detect IgG based on the PL enhancement.
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2.3. Spectra measurement
Fig. 1. Structure of morin.
This has promising applications in novel optoelectronic devices, such as microcavities, solid state lasers and flat panel displays.
The PL spectra of all samples were measured at room temperature by a Hitachi M-850 fluorescence spectrophotometer, and the absorption spectra were measured by a Shimadzu UV-240 ultraviolet (UV) – visible spectrophotometer. The Fourier transform infrared (FTIR) spectra of all samples were measured by a Nicolet Nexus 670 FTIR spectrometer (America) equipped with a Germanium attenuated total reflection (ATR) accessory, a DTGS KBr detector and a KBr beam splitter.
3. Results and discussion 2. Experimental details 2.1. AAO preparation High-purity (99.999%) aluminum (dimensions 30 10 mm, thickness 1 mm) was used as the starting material. Before anodization the aluminum was annealed at 600 jC for 6 h and then degreased ultrasonically with acetone and cleaned in 5% NaOH at 15 jC for 20 min. After that, the aluminum was electropolished in a 25:75 volume mixture of HClO4 and C2H5OH. Porous alumina membranes were prepared in a two-step anodizing process [13]. The polished alumina foil was anodized in a 0.3 mol l 1 oxalic acid (H2C2O4) electrolyte. The anodizing voltage was 40 V DC and the temperature of the electrolyte was kept constant at 10 jC. After the 3 h anodization, the specimens were immersed in a mixture of 6 wt.% H3PO4 and 1.8 wt.% H2CrO4 at 60 jC for 5 min to remove the alumina layers. The alumina foil was then anodized again for 1 h under the same anodization conditions as the first step. The AAO with different size ranges from several tens to hundreds nanometers was achieved by adjusting the height of voltage in different anodizing solutions such as sulfuric, oxalic and phosphoric acid. The morphology of the as-prepared AAO membranes was observed by a JSM-5600LV scanning electron microscope (SEM). The average diameters of AAO layer used in the present work were 50, 75 and 100 nm, respectively. 2.2. Absorption of dye and protein Morin was purchased from Beijing chemical plant. The stock solution of morin (1.0 10 2 mol l 1) was prepared by dissolving it in 95% ethanol. Human immunoglobulin (Sigma, St. Louis, MO) was directly dissolved in deionized water to prepare stock solution of 1000 Ag ml 1, and stocked at 0– 4 jC. Porous AAO membranes prepared in H2C2O4 were dipped in 30% ethanol solutions of dye and IgG with different concentrations for different soaking time. Then they were removed, flushed fully with 30% ethanol and dried.
The soaking sequence of AAO layer has some effect on the PL intensity. No emission signal was detected when the AAO film was dipped in IgG solution, then an evident PL band near 500 nm appeared following immersion in morin solution for some time, whose position is similar to that of morin– Al3 + complex. Additionally, when the AAO film was immersed in morin solution first, a weak PL band was found; after immersion into IgG solution, the PL was enhanced, but its intensity was much weaker than that of a AAO membrane dipped into the solution containing both dye and IgG directly. Based on the above phenomena, we infer that dye and dye– IgG impregnated in the nanometersized holes should be responsible for the appearance and enhancement of the PL band detected here, respectively. A subsequent cleaning treatment of AAO impregnated dye or dye – IgG was undertaken. Thoroughly rinsed with doubly distilled water or 30% ethanol, or even acetone, the PL intensity of AAO film was hardly weakened, especially for embedded morin – IgG. Only after an ultrasonic cleaning for 2 h, the PL was obviously weakened. Even after an ultrasonic treatment during more than 5 h, the PL band did not completely disappear. These results strongly suggest that dye and protein embedded in the pores of AAO membrane are not only physically adsorbed, but that some type of chemical reaction in the inner wall of AAO pores could occur. Hence, the AAO layer was immersed into solutions containing dye and IgG or not in all subsequent work. As we know, AAO itself has a blue PL band which originates from singly ionized oxygen vacancy (F+ center) in AAO membranes [14], but it does not present the PL band under the measured conditions. However, when dye or dye – IgG molecules were introduced into the holes or on the surface of AAO film, an evident PL band whose position is similar to that of morin – Al3 + complex in 30% ethanol solution appeared, and the intensity of embedded dye – protein was much higher than that of embedded dye, as shown in Fig. 2c, e and f. Moreover, the addition of IgG hardly changes the PL positions of them both but varies their intensities a little. Considering the nanometer pore size of the alumina substrate and comparing with dye or dye–
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Fig. 2. Emission spectra of (a) morin; (b) morin – IgG; (c) morin – Al3 + (excited at 450 nm); (d) Morin – Al3 + – IgG (excited at 450 nm) in 30% ethanol solutions; (e and f) AAO after absorbing in (a) and (b), respectively. Unless otherwise indicated: dye concentration, 2 10 4 mol l 1; IgG concentration, 120 Ag ml 1; Al3 +, 2 10 4 mol l 1; excitation wavelength, 410 nm; immersion time, 0.5 h; pore diameter, 100 nm.
IgG solution in 30% ethanol solution, we believe that the blue shift is attributed to the aggregates of dye or dye –IgG repressed in the nanometer scale pores. In solution, dimers and multimers form more easily due to strong interaction between the molecules, which leads to a decrease in the gap of the molecules and enhancement of the interaction of the electrons and phonons [15], so a red shift in the PL is observed. However, when the molecules are buried in the nanometer-sized holes, the interaction between them is reduced and the shift moves towards longer wavelength. Fig. 3 presents the absorption spectra of AAO films and its corresponding soaking solutions. As shown in curve (c) of Fig. 3B, no characteristic absorption of AAO membrane near 340 nm was found. However, when dye or dye –IgG molecules were adsorbed on the inner wall of cylindrical pores, the absorption peak around 340 nm appeared, as shown in curves (d) and (e) of Fig. 3B. Similar results, as shown in Fig. 3A, were observed from the liquid samples of dye and dye– IgG. This indicates that the PL band near 500 nm does not originate from the AAO but from dye or dye– IgG immersed in the AAO pores. The FTIR measurements further proved the presence of morin or morin – IgG in the nanometer-sized holes, as presented in Fig. 4. Obviously, no characteristic absorption band of AAO film by itself was found in the 1400 – 1800 cm 1 region, as shown in curve (a) of Fig. 4C. Compared with the absorption spectrum of pure morin (see Fig. 4A), it was found that the absorption positions of embedded morin, which orginate from the stretching vibration of the aromatic rings, shift obviously from 1448 to 1482 cm 1 and from 1507 to 1593 cm 1, respectively. Meanwhile, the CMO and CMC groups of embedded morin give rise to two relatively weak bands at 1712 and 1663 cm 1, as presented in curve (b) of Fig. 4C. They both show a higher-frequency shift relatively to that of pure morin itself at 1652 and 1566 cm 1, respectively, being assigned from its standard spec-
trum [16]. Fig. 4B and curve (c) of Fig. 4C present the characteristic absorption of pure and embedded IgG, which originates from the strong CMO stretch band of amide group (amide 1) and the band that involves both CUN stretch and CUNUH in-plane bend in the stretch-bend mode (amide II), respectively [17]. It was found that the frequencies of the amide I and II bands shift slightly from 1646 to 1656 cm 1 and from 1535 to 1544 cm 1, respectively. When molecules of dye and IgG are both impregnated into the pores, the absorption positions of embedded dye and IgG shift slightly compared with those of embedded dye or IgG. This indicated that molecules of both dye and IgG are buried in the holes of AAO film, and some type of interaction between them might occur (see curve (d) of Fig. 4C). Fig. 5A shows the pore size of AAO membrane has great effect on the PL position and intensity. Obviously, the PL intensity increased with increasing pore size of AAO membranes. Meanwhile, the PL band shifted slightly towards the long-wavelength region, which is attributed to the aggregation of the molecules in the AAO holes. The
Fig. 3. UV – visible absorption spectra of (A) ethanol solution: (a) morin and (b) morin – IgG in 30% ethanol solutions; and (B) AAO film: (c) as prepared; (d and e) AAO film after absorbing in morin and morin – IgG in 30% ethanol solution, respectively. Unless otherwise indicated: dye concentration, 2 10 4 mol l 1; IgG concentration, 120 Ag ml 1; immersion time, 0.5 h; pore diameter, 100 nm.
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Fig. 4. FTIR spectra of (A) morin; (B) IgG; (C) AAO film: (a) as prepared; (b, c and d) AAO film after absorbing in dye, IgG and dye – IgG 30% ethanol solution, respectively. Unless otherwise indicated: dye concentration, 2 10 4 mol l 1; IgG concentration, 120 Ag ml 1; immersion time, 0.5 h; pore diameter, 100 nm.
molecules are more easily packed in a larger pore, so dimers or multimers are more easily formed than in a small pore [5]. In other words, the PL of molecules in an AAO hole resembles that of monomers, but in a larger hole, the spectrum is close to that of molecules in a high-concentration solution, thus, the PL shifts to longer wavelength. Hence, the pore size of AAO membrane was about 100 nm in subsequent experiments. It can be seen in Fig. 5B that a distinct decrease in PL intensity with longer absorption
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time, so 30 min of soaking time was chosen in subsequent work. The dependence of PL enhancement on dye or IgG concentration in the soaking solutions into which AAO film was dipped is shown in Fig. 5C and D. The maximum PL enhancement reached at the dye concentration 2 10 4 mol l 1 (see Fig. 5C). As for the IgG introduction, the PL intensity increased with increasing IgG concentration first, reached a maximum at the IgG concentration of 120 Ag ml 1 and then decreased (see Fig. 5D). It is well known that AAO membrane obtained by anodization consists primarily of disordered Al2O3, in which there is a large number of nanometer-sized cylindrical pores. So it may present strong surface effect [18] due to its colossal specific area. That is, there must be numerous aluminum atoms exposed on the inside surface of the AAO pores, which are characterized by their high reactivity and instability for being under-coordinated and having high surface energy. Therefore, when dye molecules are introduced into the cylindrical AAO pores, they may react with the remaining aluminum on the inside surface of holes to form the morin – Al complex, which results in change in the surrounding of dye molecules. However, compared with solution, the dye molecules are more confined in AAO, and therefore the binding of an aluminum to the dye might be restricted to only some certain directions; thus, full-coordinated complex cannot form as freely as in solution and the PL intensity is much weaker than that of morin –Al3 + in the liquid phase. Based on the formation of morin – Al complex with the inner wall of AAO pores involved, it was considered that the observed PL enhancement was attributed to the presence of IgG in the AAO pores. In a pH 4.5 soaking solution, the tight conformation of IgG (pI = 5.8 – 6.6) can be readily converted to the highly charged unfolded state. As a result, almost all hydrophobic groups of IgG molecule in a dispersed state unfolds, these molecules with an expandable form can accept a larger number of protons relative to the same well-known IgG. When the negative dye probe (pk1 = 1) [19] was close to IgG, dye binds to protonated amine groups of amino acid residues in the polypeptide chain of IgG to form the dye –HSA aggregate due to the electrostatic attraction. Meanwhile, the aggregation was stabilized through the combination of hydrophobic groups of dye molecule and hydrophobic amine acid residues. According to Beer’s Law, the molar absorption coefficient of color material is proportional to its effective absorption area. When the dye – IgG aggregate form, the effective absorption area of dye – IgG increase largely relatively to that of morin [20]. So it was seen in Fig. 3A that the absorption intensity of dye –IgG was a little bigger than that of dye. When the morin– IgG aggregate was impregnated in a nanometer-sized hole, they may react with the remaining aluminum on the inside surface of AAO pores to form an Al – morin –IgG complex, and therefore an orderly, stable and spatial system containing of bio-macromolecule, dye
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Fig. 5. PL spectra of AAO after absorbing in dye and IgG ethanol (30%) solution as a function of (A) pore diameter (nm); (B) immersion time (h); (C) dye concentration ( 10 5 mol l 1); and (D) IgG concentration (Ag ml 1). Unless otherwise indicated: dye concentration, 2 10 4 mol l 1; IgG concentration, 200 Ag ml 1; excitation wavelength, 410 nm; immersion time, 0.5 h; pore diameter, 100 nm.
and the inner wall of pores involved is formed, which can show much higher luminescent efficiency relative to disordered dye –Al complex in a nanometer-sized hole, thus, a PL enhancement with the PL position unchanged can be observed. Without protein molecule, no ordered spatial system can exist. This may be the reason why the PL intensity of embedded dye can be enhanced by the introduction of protein.
the proposed approach is very promising for the preparation of novel optoelectronic devices or luminescent material with biomacromolecule involved.
Acknowledgements This work was supported by the National Natural Scientific Foundation of China (No. 20275014) and Visiting Scholar Foundation of Key Lab. in Lanzhou University.
4. Conclusion In this study, the PL spectra of morin and human immunoglobulin absorbed in the pores of AAO film were investigated. It was found that their PL bands are similar to that of dye – Al3 + complex, which may be due to the interaction of morin with the remaining aluminum atoms exposed on the inside surface of AAO holes. Furthermore, the PL intensity of embedded dye can be enhanced greatly by the addition of IgG. We inferred that the PL enhancement might be due to the coexistence of dye and IgG in the holes, which has been proven by UV and FITR measurements. Based on this, a novel for the luminescence is proposed. It was also found that the PL intensity increased with pore sizes increased. Although more work is still needed to understand the underlying mechanisms, we believe that
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[16] Standard Infrared Grating Spectra 35 35045K (Sadtler Research Laboratories, USA 1975). [17] K. Rahmelow, W. Hubner, Anal. Biochem. 241 (1996) 5. [18] R.E. Cavicchi, R.H. Silsbee, Phys. Rev. Lett. 52 (1984) 1453. [19] M.H. Fletcher, Anal. Chem. 37 (1965) 550. [20] X.Y. Zhang, F. Chen, C.H. Peng, H.Z. Hua, S.Y. Tong, Principle of Chemical Analysis (Series of Analytical Chemistry), vol. 1(1), Science Press, Beijing, 1991, p. 379.
Photoluminescence behaviors of morin–human immunoglobulin on porous anodized aluminum oxide films R.P. Jia a, Y. Shen a, H.Q. Luo a, X.G. Chen a,*, Z.D. Hu a, D.S. Xue b a
b
Department of Chemistry, Lanzhou University, Lanzhou 730000, PR China Key Lab for Magnetism and Magnetic Materials of MOE, Lanzhou University, Lanzhou 730000, PR China Received 8 October 2003; received in revised form 25 May 2004; accepted 9 June 2004 Available online 19 July 2004
Abstract For the first time, the photoluminescence (PL) spectra of porous anodized aluminum oxide (AAO) films impregnated with essentially non-fluorescent 3, 5, 7, 2V, 4V-pentahydroxyflavone (morin) and morin – human immunoglobulin (IgG) were investigated and compared with those of liquid solutions. It was found that their PL band positions are similar to that of morin – Al3 + in ethanol solution, and the PL intensity of embedded morin alone can be enhanced greatly by the introduction of human immunoglobulin. We infer that the appearance of the PL band detected here is due to the formation of morin – Al complex in the holes of AAO with the inner wall involved, and a likely luminescent mechanism is proposed to elucidate the PL enhancement phenomena due to the coexistence of morin and IgG in the AAO pores, which is confirmed by ultraviolet (UV) – visible and Fourier transform infrared (FTIR) measurements. Moreover, it is also found that the PL intensity increases with the pore size. D 2004 Elsevier B.V. All rights reserved. Keywords: Photoluminescence; Morin; IgG; AAO film
1. Introduction Porous anodized aluminum oxide (AAO), a typical selfordered nano-channel material formed by anodizing Al in an appropriate acid solution, has recently stimulated considerable interest as a key material for the fabrication of nanometer-scale structures [1 – 3]. The structure of AAO is described as a closely packed array of columnar cells, each containing a central pore of which the size and interval can be controlled by changing the forming conditions [4]. In recent years, a few attempts have been focused on the optical properties of luminescent substances embedded in the pores of AAO layer, which have shown promising applications in novel optoelectronic devices, such as microcavities, solid state lasers and so on [5,6]. However, as far as we know, there is no report on the luminescent properties and corresponding luminescent mechanisms of small organ-
* Corresponding author. Fax: +86-9318912582. E-mail addresses: [email protected], [email protected] (X.G. Chen). 0040-6090/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.tsf.2004.06.113
ic dye molecule and bio-macromolecule impregnated in the pores of AAO films. 3, 5, 7, 2V, 4V-pentahydroxyflavone (morin, its structure is shown in Fig. 1), a kind of O, O-donating chelating reagent, is only weakly fluorescent by itself but forms highly fluorescent complex with Al3 +. Based on this, a sensitive method for the determination of aluminum was developed; the detection limit can be reached to the parts-per-billion range [7,8]. Moreover, this dye also has been used as a probe for other metal ions [9,10] and some bio-macromolecule, such as protein [11] and nucleic acid [12] by the liquid phase. The aim of this work is to draw attention to a complementary approach that utilizes AAO technology to develop the luminescence of small organic molecules and bio-macromolecules in a nanometer-sized structure. Based on the above-mentioned remarks, in this paper, we investigated for the first time the photoluminescence (PL) properties of AAO films immersed in solutions containing dye and human immunoglobulin (IgG) or not, and a possible luminescence mechanism is proposed to explain the PL enhancement. We hope that the proposed approach would be useful to detect IgG based on the PL enhancement.
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2.3. Spectra measurement
Fig. 1. Structure of morin.
This has promising applications in novel optoelectronic devices, such as microcavities, solid state lasers and flat panel displays.
The PL spectra of all samples were measured at room temperature by a Hitachi M-850 fluorescence spectrophotometer, and the absorption spectra were measured by a Shimadzu UV-240 ultraviolet (UV) – visible spectrophotometer. The Fourier transform infrared (FTIR) spectra of all samples were measured by a Nicolet Nexus 670 FTIR spectrometer (America) equipped with a Germanium attenuated total reflection (ATR) accessory, a DTGS KBr detector and a KBr beam splitter.
3. Results and discussion 2. Experimental details 2.1. AAO preparation High-purity (99.999%) aluminum (dimensions 30 10 mm, thickness 1 mm) was used as the starting material. Before anodization the aluminum was annealed at 600 jC for 6 h and then degreased ultrasonically with acetone and cleaned in 5% NaOH at 15 jC for 20 min. After that, the aluminum was electropolished in a 25:75 volume mixture of HClO4 and C2H5OH. Porous alumina membranes were prepared in a two-step anodizing process [13]. The polished alumina foil was anodized in a 0.3 mol l 1 oxalic acid (H2C2O4) electrolyte. The anodizing voltage was 40 V DC and the temperature of the electrolyte was kept constant at 10 jC. After the 3 h anodization, the specimens were immersed in a mixture of 6 wt.% H3PO4 and 1.8 wt.% H2CrO4 at 60 jC for 5 min to remove the alumina layers. The alumina foil was then anodized again for 1 h under the same anodization conditions as the first step. The AAO with different size ranges from several tens to hundreds nanometers was achieved by adjusting the height of voltage in different anodizing solutions such as sulfuric, oxalic and phosphoric acid. The morphology of the as-prepared AAO membranes was observed by a JSM-5600LV scanning electron microscope (SEM). The average diameters of AAO layer used in the present work were 50, 75 and 100 nm, respectively. 2.2. Absorption of dye and protein Morin was purchased from Beijing chemical plant. The stock solution of morin (1.0 10 2 mol l 1) was prepared by dissolving it in 95% ethanol. Human immunoglobulin (Sigma, St. Louis, MO) was directly dissolved in deionized water to prepare stock solution of 1000 Ag ml 1, and stocked at 0– 4 jC. Porous AAO membranes prepared in H2C2O4 were dipped in 30% ethanol solutions of dye and IgG with different concentrations for different soaking time. Then they were removed, flushed fully with 30% ethanol and dried.
The soaking sequence of AAO layer has some effect on the PL intensity. No emission signal was detected when the AAO film was dipped in IgG solution, then an evident PL band near 500 nm appeared following immersion in morin solution for some time, whose position is similar to that of morin– Al3 + complex. Additionally, when the AAO film was immersed in morin solution first, a weak PL band was found; after immersion into IgG solution, the PL was enhanced, but its intensity was much weaker than that of a AAO membrane dipped into the solution containing both dye and IgG directly. Based on the above phenomena, we infer that dye and dye– IgG impregnated in the nanometersized holes should be responsible for the appearance and enhancement of the PL band detected here, respectively. A subsequent cleaning treatment of AAO impregnated dye or dye – IgG was undertaken. Thoroughly rinsed with doubly distilled water or 30% ethanol, or even acetone, the PL intensity of AAO film was hardly weakened, especially for embedded morin – IgG. Only after an ultrasonic cleaning for 2 h, the PL was obviously weakened. Even after an ultrasonic treatment during more than 5 h, the PL band did not completely disappear. These results strongly suggest that dye and protein embedded in the pores of AAO membrane are not only physically adsorbed, but that some type of chemical reaction in the inner wall of AAO pores could occur. Hence, the AAO layer was immersed into solutions containing dye and IgG or not in all subsequent work. As we know, AAO itself has a blue PL band which originates from singly ionized oxygen vacancy (F+ center) in AAO membranes [14], but it does not present the PL band under the measured conditions. However, when dye or dye – IgG molecules were introduced into the holes or on the surface of AAO film, an evident PL band whose position is similar to that of morin – Al3 + complex in 30% ethanol solution appeared, and the intensity of embedded dye – protein was much higher than that of embedded dye, as shown in Fig. 2c, e and f. Moreover, the addition of IgG hardly changes the PL positions of them both but varies their intensities a little. Considering the nanometer pore size of the alumina substrate and comparing with dye or dye–
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Fig. 2. Emission spectra of (a) morin; (b) morin – IgG; (c) morin – Al3 + (excited at 450 nm); (d) Morin – Al3 + – IgG (excited at 450 nm) in 30% ethanol solutions; (e and f) AAO after absorbing in (a) and (b), respectively. Unless otherwise indicated: dye concentration, 2 10 4 mol l 1; IgG concentration, 120 Ag ml 1; Al3 +, 2 10 4 mol l 1; excitation wavelength, 410 nm; immersion time, 0.5 h; pore diameter, 100 nm.
IgG solution in 30% ethanol solution, we believe that the blue shift is attributed to the aggregates of dye or dye –IgG repressed in the nanometer scale pores. In solution, dimers and multimers form more easily due to strong interaction between the molecules, which leads to a decrease in the gap of the molecules and enhancement of the interaction of the electrons and phonons [15], so a red shift in the PL is observed. However, when the molecules are buried in the nanometer-sized holes, the interaction between them is reduced and the shift moves towards longer wavelength. Fig. 3 presents the absorption spectra of AAO films and its corresponding soaking solutions. As shown in curve (c) of Fig. 3B, no characteristic absorption of AAO membrane near 340 nm was found. However, when dye or dye –IgG molecules were adsorbed on the inner wall of cylindrical pores, the absorption peak around 340 nm appeared, as shown in curves (d) and (e) of Fig. 3B. Similar results, as shown in Fig. 3A, were observed from the liquid samples of dye and dye– IgG. This indicates that the PL band near 500 nm does not originate from the AAO but from dye or dye– IgG immersed in the AAO pores. The FTIR measurements further proved the presence of morin or morin – IgG in the nanometer-sized holes, as presented in Fig. 4. Obviously, no characteristic absorption band of AAO film by itself was found in the 1400 – 1800 cm 1 region, as shown in curve (a) of Fig. 4C. Compared with the absorption spectrum of pure morin (see Fig. 4A), it was found that the absorption positions of embedded morin, which orginate from the stretching vibration of the aromatic rings, shift obviously from 1448 to 1482 cm 1 and from 1507 to 1593 cm 1, respectively. Meanwhile, the CMO and CMC groups of embedded morin give rise to two relatively weak bands at 1712 and 1663 cm 1, as presented in curve (b) of Fig. 4C. They both show a higher-frequency shift relatively to that of pure morin itself at 1652 and 1566 cm 1, respectively, being assigned from its standard spec-
trum [16]. Fig. 4B and curve (c) of Fig. 4C present the characteristic absorption of pure and embedded IgG, which originates from the strong CMO stretch band of amide group (amide 1) and the band that involves both CUN stretch and CUNUH in-plane bend in the stretch-bend mode (amide II), respectively [17]. It was found that the frequencies of the amide I and II bands shift slightly from 1646 to 1656 cm 1 and from 1535 to 1544 cm 1, respectively. When molecules of dye and IgG are both impregnated into the pores, the absorption positions of embedded dye and IgG shift slightly compared with those of embedded dye or IgG. This indicated that molecules of both dye and IgG are buried in the holes of AAO film, and some type of interaction between them might occur (see curve (d) of Fig. 4C). Fig. 5A shows the pore size of AAO membrane has great effect on the PL position and intensity. Obviously, the PL intensity increased with increasing pore size of AAO membranes. Meanwhile, the PL band shifted slightly towards the long-wavelength region, which is attributed to the aggregation of the molecules in the AAO holes. The
Fig. 3. UV – visible absorption spectra of (A) ethanol solution: (a) morin and (b) morin – IgG in 30% ethanol solutions; and (B) AAO film: (c) as prepared; (d and e) AAO film after absorbing in morin and morin – IgG in 30% ethanol solution, respectively. Unless otherwise indicated: dye concentration, 2 10 4 mol l 1; IgG concentration, 120 Ag ml 1; immersion time, 0.5 h; pore diameter, 100 nm.
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Fig. 4. FTIR spectra of (A) morin; (B) IgG; (C) AAO film: (a) as prepared; (b, c and d) AAO film after absorbing in dye, IgG and dye – IgG 30% ethanol solution, respectively. Unless otherwise indicated: dye concentration, 2 10 4 mol l 1; IgG concentration, 120 Ag ml 1; immersion time, 0.5 h; pore diameter, 100 nm.
molecules are more easily packed in a larger pore, so dimers or multimers are more easily formed than in a small pore [5]. In other words, the PL of molecules in an AAO hole resembles that of monomers, but in a larger hole, the spectrum is close to that of molecules in a high-concentration solution, thus, the PL shifts to longer wavelength. Hence, the pore size of AAO membrane was about 100 nm in subsequent experiments. It can be seen in Fig. 5B that a distinct decrease in PL intensity with longer absorption
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time, so 30 min of soaking time was chosen in subsequent work. The dependence of PL enhancement on dye or IgG concentration in the soaking solutions into which AAO film was dipped is shown in Fig. 5C and D. The maximum PL enhancement reached at the dye concentration 2 10 4 mol l 1 (see Fig. 5C). As for the IgG introduction, the PL intensity increased with increasing IgG concentration first, reached a maximum at the IgG concentration of 120 Ag ml 1 and then decreased (see Fig. 5D). It is well known that AAO membrane obtained by anodization consists primarily of disordered Al2O3, in which there is a large number of nanometer-sized cylindrical pores. So it may present strong surface effect [18] due to its colossal specific area. That is, there must be numerous aluminum atoms exposed on the inside surface of the AAO pores, which are characterized by their high reactivity and instability for being under-coordinated and having high surface energy. Therefore, when dye molecules are introduced into the cylindrical AAO pores, they may react with the remaining aluminum on the inside surface of holes to form the morin – Al complex, which results in change in the surrounding of dye molecules. However, compared with solution, the dye molecules are more confined in AAO, and therefore the binding of an aluminum to the dye might be restricted to only some certain directions; thus, full-coordinated complex cannot form as freely as in solution and the PL intensity is much weaker than that of morin –Al3 + in the liquid phase. Based on the formation of morin – Al complex with the inner wall of AAO pores involved, it was considered that the observed PL enhancement was attributed to the presence of IgG in the AAO pores. In a pH 4.5 soaking solution, the tight conformation of IgG (pI = 5.8 – 6.6) can be readily converted to the highly charged unfolded state. As a result, almost all hydrophobic groups of IgG molecule in a dispersed state unfolds, these molecules with an expandable form can accept a larger number of protons relative to the same well-known IgG. When the negative dye probe (pk1 = 1) [19] was close to IgG, dye binds to protonated amine groups of amino acid residues in the polypeptide chain of IgG to form the dye –HSA aggregate due to the electrostatic attraction. Meanwhile, the aggregation was stabilized through the combination of hydrophobic groups of dye molecule and hydrophobic amine acid residues. According to Beer’s Law, the molar absorption coefficient of color material is proportional to its effective absorption area. When the dye – IgG aggregate form, the effective absorption area of dye – IgG increase largely relatively to that of morin [20]. So it was seen in Fig. 3A that the absorption intensity of dye –IgG was a little bigger than that of dye. When the morin– IgG aggregate was impregnated in a nanometer-sized hole, they may react with the remaining aluminum on the inside surface of AAO pores to form an Al – morin –IgG complex, and therefore an orderly, stable and spatial system containing of bio-macromolecule, dye
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Fig. 5. PL spectra of AAO after absorbing in dye and IgG ethanol (30%) solution as a function of (A) pore diameter (nm); (B) immersion time (h); (C) dye concentration ( 10 5 mol l 1); and (D) IgG concentration (Ag ml 1). Unless otherwise indicated: dye concentration, 2 10 4 mol l 1; IgG concentration, 200 Ag ml 1; excitation wavelength, 410 nm; immersion time, 0.5 h; pore diameter, 100 nm.
and the inner wall of pores involved is formed, which can show much higher luminescent efficiency relative to disordered dye –Al complex in a nanometer-sized hole, thus, a PL enhancement with the PL position unchanged can be observed. Without protein molecule, no ordered spatial system can exist. This may be the reason why the PL intensity of embedded dye can be enhanced by the introduction of protein.
the proposed approach is very promising for the preparation of novel optoelectronic devices or luminescent material with biomacromolecule involved.
Acknowledgements This work was supported by the National Natural Scientific Foundation of China (No. 20275014) and Visiting Scholar Foundation of Key Lab. in Lanzhou University.
4. Conclusion In this study, the PL spectra of morin and human immunoglobulin absorbed in the pores of AAO film were investigated. It was found that their PL bands are similar to that of dye – Al3 + complex, which may be due to the interaction of morin with the remaining aluminum atoms exposed on the inside surface of AAO holes. Furthermore, the PL intensity of embedded dye can be enhanced greatly by the addition of IgG. We inferred that the PL enhancement might be due to the coexistence of dye and IgG in the holes, which has been proven by UV and FITR measurements. Based on this, a novel for the luminescence is proposed. It was also found that the PL intensity increased with pore sizes increased. Although more work is still needed to understand the underlying mechanisms, we believe that
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