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particle-based immunofluorescence assay: Synthesis, characterization and application
Journal of Photochemistry and Photobiology B: Biology 94 (2009) 45–50
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / j p h o t o b i o l
Quantum dots/particle-based immunofluorescence assay: Synthesis, characterization and application Bingbo Zhang a, Xiaofei Liang a, Lijuan Hao a, Jing Cheng a, Xiaoqun Gong a, Xuhui Liu b, Guiping Ma b, Jin Chang a,* a b
Institute of Nanobiotechnology, School of Materials Science and Engineering, Tianjin University, No. 92 Weijin Road, Nankai District, Tianjin 300072, China Center of Technology, Beijing Entr-exit Inspection and Quarantine Bureau, Beijing 100029, China
a r t i c l e
i n f o
Article history: Received 2 May 2008 Received in revised form 14 September 2008 Accepted 29 September 2008 Available online xxxx Keywords: Quantum dots Polystyrene microspheres Magnetic Fluorescent Immunofluorescence assays
a b s t r a c t Recently, it has been proved that quantum dots (QDs) hold the potential to be used in the bioanalysis as fluorescent probes for their many unique optical properties. In this paper, immunofluorescence assay, an integration of particle-based immunoassays and fluorescent QD-probes, was constructed. Firstly, high qual ity CdSe/ZnS QDs were prepared. Then after being water-solubilized by amphiphilic polymer based on selfassembling, the QDs were labeled by immunoglobulin G (IgG) antibody. At the same time, both carboxylpolystyrene (PS) and magnetic carboxyl-PS microspheres were prepared and coated by antigens. The antigen sensitized PS microspheres were specifically captured by the QD–IgG bioconjugates based on the antibodyantigen reaction, which was confirmed by the immunofluorescence test in vitro. The sensitivity of current assay was tested by sandwich immunofluorescence assay using human alpha fetoprotein (AFP) as antigen model. The detection limit of AFP antigen is 4.9 ng/mL. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Particle capture enzyme-linked immunosorbent assay (ELISA) tests, based on the very specific interaction of antibody and anti gen, have been in common use, since latex particles or micro spheres were first used in medical diagnostic application as latex agglutination test (LAT) in the late 1950s [1]. Nowadays, there have been many innovative improvements based on the latex particles including colored particles [2], special devices for results inter pretation [3–5], magnetic particles [6], and integration of parti cles and fluorescent report probes [4,7]. Magnetic particles can be used to help pull things out of solution more quickly, which offers an easy solid–liquid separ ation [8]. Integrated with report probes, especially fluorescent probes, particles-based immunoassays have attracted great attentions from biologist, chemist, and materials researchers. The fluorescent probes used in immunofluorescence assays are usually conventional fluorophores/dye [7]. Those fluoro phores are not suitable for extended periods of observations using fluorescent and confocal microscopy because organic fluorophores tend to quench rapidly [9]. Furthermore, it is sometimes dif ficult or impossible to record fine fluorescent images while the organic dye probes fade in the course of adjusting the focus. In contrast, QDs, also called inorganic nanocrystals [10–14], are stabilized over a far longer exposure-time to light and can emit a fluorescence of high * Corresponding author. Tel./fax: +86 22 27401821. E-mail address: jin[email protected] (J. Chang). 1011-1344/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2008.09.008
luminosity at an almost equivalent condition as the conventional organic dye probes. Besides that, QDs have many other advanta ges over traditional organic dyes: tunable fluorescent wavelength by raw materials and size, broad excitation spectra, sharp and symmetrical fluorescent peak, large Stokes shift, and differentsized QDs can be excited simultaneously by a single wavelength [15]. Problems with organic fluorophores include narrow excita tion bands and broad emission spectra, which makes detection of multiple color emitting probes dif ficult due to spectral over lap. With improvement of preparation of water-soluble QDs, they have shown increasing promising applications in biological fields, especially as fluorescent probes for biological markers, biological detection and biological imaging area [16–18]. In this paper, we described a novel method, integration of particles and QDs, for preliminary biodetection. Firstly the carboxyl-PS and magnetic carboxyl-PS microspheres were covalently attached by antigen/ antibody. Then the sensitized microspheres acted to magnify the antibody-antigen reaction when they were mixed with antibodylabeled QD-probes. 2. Materials and methods 2.1. Materials and apparatus Selenium powder (100 mesh, 99.99%, Aldrich), Cadmium oxide (CdO, 99.5%, Aldrich), Zinc oxide (ZnO, 99.9%, Sigma), Sulphur (99.98%, Aldrich), Tri-n-butylp hosp hine (TBP, 90%,TCI, Japan),
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B. Zhang et al. / Journal of Photochemistry and Photobiology B: Biology 94 (2009) 45–50
Tri-n-octylphosphine oxide (TOPO, 90%, Aldrich), Octadecylamine (ODA, 90%, ACROS), 1-Octadecene (ODE, 90%, ACROS), Oleic acid (OA, 90%, Aldrich), Octylamine (99%, Aldrich). Carboxyl- PS micro spheres (Tianjin BaseLine ChromTech Research Centre, 7–8 lm), 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC, GL Biochem (Shanghai) Ltd.), Human IgG: purified total IgG from normal human serum, in which heavy chain is 50.000 Da, light chain is 25.000 Da. Goat anti-human IgG: pure human total IgG immune against goat until 1:100000 (ELISA), Goat anti-mouse IgG: pure mouse total IgG immune against goat until 1:100000 (ELISA),above supplied by BEIJING DINGGUO BIOTECHNOLOGY CO.LTD. Human alpha fetoprotein (AFP) antigen, mouse monoclo nal antibody to human AFP (Clone: A3 for PS microspheres coat ing, Clone: D2 for QDs labeling), supplied by Beijing Keyuebio BiotechCo. Ltd. chloroform, ethanol, dichloromethane and argon (oxygen free) were obtained from local suppliers. All chemicals were used without further purification. Ultraviolet–visible (UV–vis) absorption spectra were recorded on UV-2450 spectrophotometer. Photoluminescence (PL) mea surement was performed at room temperat ure using an F-4500 (HITACHI) spectrophotometer. QDs and hydrophobic ferric oxide nanocrystals were visualized using a Tecnai G2 F20 transmission electron microscope (TEM) operating at an accelerat ion voltage of 200 kV. The magnetic property of PS microspheres was analyzed at Vibrating Samples Magetometer (VSM), LDJ9600-1. Immu nofluor escence assay were examined under an OLYMPUS.BX51 fluorescence microscope equipped with an OLYMPUS MICRO DP 70 camera. 2.2. Synthesis of water-soluble amphiphilic triblock copolymer-capped CdSe/ZnS QDs CdSe/ZnS QDs were synthesized according to the previously reported procedure [19]. However, amphiphilic triblock copolymer was selected as the surface capping reagent to form water-soluble QDs for labeling IgG. Triblock copolymer from Sigma consists of a polybutylacrylate segment, a polyethylacrylate segment and a poly methacrylic acid segment. At a molecular weight of ca.100,000 Da, for better encapsulation of QDs, about 25% of the free carboxylic acid groups were derivatized with octylamine (a hydrophobic side chain). CdSe/ZnS QDs and amphiphilic triblock polymer (The mol ratio of QDs to polymer was 1:10) were dissolved in chloroform/ ethanol solvent mixture (3:1, v/v) and stirred for several hours for chloroform evaporation. After evapor ation of chloroform, the encapsulated dots were suspended in PBS, sonicated for 3 min and then purified by gel filtration (sepharose 6 fast flow, GE healthcare life sciences).
as coupling agent in the PBS buffer for 2.0 h. The mol ratios ofQDs/ IgG/EDC were in 1:15:4000. The final QDs–IgG bioconjugates were purified by ultracentrifugation, and then dissolved in the PBS (0.01 M, pH 7.2, 0.5%BSA). Highly carboxylated PS microspheres, including ferric oxide doped carboxyl-PS microspheres, were both coated with human IgG via the covalent bonds between human IgG molecules and microspheres. The solutions of human IgG (1.0 mg/mL, 0.1 mL), EDC (10 mg) and microspheres (10 mg/mL, 1.0 mL) were gently mixed in the PBS buffer for two hours at room temperature using an orbital shaker followed by storage at 4 °C. The above coupling protocols are feas ib le for the micros pheres coati ng with mouse anti-AFP McAb, and QDs labeling with mouse anti-AFP McAb. 2.5. Immunofluorescence assay The immunofluorescence assay for the positive and negative control experiments were both carried out in the same manner. QD-goat anti-human IgG bioconjugates were used as the positive control, while QD-goat anti-mouse IgG bioconjugates as the neg ative control. The human IgG-coated PS microspheres (2.0 mg/mL, 1.0 mL) were firstly blocked for 30 min at room temperature in the PBS–BSA buffer (0.01 M, pH 7.2, 0.5%BSA), then gently mixed with QD-goat anti-human IgG bioconjugates (16 nmol/L, 0.1 mL) and QD-goat anti-mouse IgG bioconjugates (16 nmol/L, 0.1 mL) sep arately for 45 min at room temperature, using an orbital shaker. After antibody-antigen reaction, centrifugations/PBS-T (0.01 M, pH 7.2, 0.05% Tween) redispersion procedures were applied to separate the unbound QD–IgG bioconjugates, since the PS micro spheres can be centrifugated down at 4000 rpm/1 min, while unboundQD–IgG bioconjugates still suspended. Finally the micro spheres were examined under an OLYMPUS.BX51 fluorescence microscope equipped with an OLYMPUS MICRO DP 70 camera and a broad band light source (ultraviolet 330–385 nm). The illumi nation light was from an O-LH100HG 100 W mercury lamp with Automatic exposure control. The sandwich assay was used to test the detection limit of AFP antigen. The mouse anti-AFP McAb (Clone: A3) coated PS micro spheres and different concentration of AFP antigen were mixed and incubated on a shaker for 1 hour, and then wash three times to remove the unbound antigens. The PS microspheres/AFP antigen complexes were obtained. Then QDs-mouse anti-AFP McAb(Clone: D2) bioconjugates were added. The reaction system was incubated with gentle shaking for another 1 h and followed by centrifuga tion. The sediments were resuspended in PBS buffer for following study. 3. Results and discussion
2.3. Preparation of ferric oxide doped carboxyl-PS microspheres Hydrophobic ferric oxide was synthesized according to a pre viously reported protocol with minor modifications [20]. Embed ding of PS beads was accomplished by doping a certain amount of ferric oxide into carboxyl-PS microspheres. Firstly the micro spheres (5.0 mg) were swelled in a solvent mixture containing chloroform and butanol (5:95, v/v) to improve the capability of absorbing ferric oxide in a centrifugal tube. Then 0.1 mg of ferric oxide dissolved in chloroform solvent was added into the tube and stirred for 2–4 h until the ferric oxide penetrate into microspheres adequately. Finally, the microspheres were washed with ethanol. 2.4. Water-soluble QDs and carboxyl-PS microspheres labeling with IgGs The as-prepared water-soluble QDs were reacted with goat anti-human IgG and goat anti-mouse IgG, respectively using EDC
The dispersion and particle sizes of polymer modified CdSe/ZnS QDs and hydrophobic ferric oxide nanocrystals were evaluated by a transmission electron microscopy (TEM) as shown in Fig. 1. The particles of polymer modified QDs were well dispersive without obvious aggregation due to electrostatic repulsion from the car boxylic groups on the surface of each QD [21]. TEM analysis also showed the prepared hydrophobic ferric oxide nanocrystals were monodisperse. Fig. 2a was the UV–visible absorption spectra of the polymer modified CdSe/ZnS QDs. The spectra showed that QDs had a broad and continuous absorption, allowing excitation by a wide range of wavelengths. And it kept the original optical property after water solubilization. Fluorescence emission spectra in Fig. 2b showed peaks both narrow and symmetric with no tailings. The red-shift of peak from 604 nm to 611 nm after water solubilization proce dure indicates that the surface environment of CdSe/ZnS changed from oil phase to water phase [22,23]. And the full width at half
B. Zhang et al. / Journal of Photochemistry and Photobiology B: Biology 94 (2009) 45–50
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Fig. 1. TEM image of polymer modified CdSe/ZnS QDs.
axim m um (FWHM) of fluorescence emission peaks broaden slightly from 27 nm to 29 nm. Planar assays based on flat carriers (glass sides, flat-bottomed ELISA plate, et al.) are among the most intensively investigated for bioassays [24]. However, suspension array based on particles is more promising since its inherent and unique characteristics. The high binding capacity of three-dimensional microspheres makes the suspension array a very sensitive platform for immunofluores cence assays. One of the most used carriers in suspension array is particles or beads. The encoded particles have great potentials for bioapplication, for example, being integrated with a flow cyto metric system [25], or Luminex flow analyzer [26] to make a highthroughput screening. Carboxyl-PS microspheres, which were easily recognizable with the optical microscopy, were used in this paper to be the solid supports for immobility of antigen via the covalent attachment. Human IgG were tagged onto the surface of microspheres using EDC as coupling agents for both positive and negative control experiments. Herein, fluorescent report QDprobes were used instead of the traditional dye molecule-probes. The QD-anti-human IgG bioconjugates were used as the positive probes, while the QD-anti-mouse IgG bioconjugates were used as the negative probes. Fig. 3a/b shows the immunofluorescence images of human IgG sensitized PS microspheres treated with QDprobes. The images showed that the fluorescence on the positive control microspheres was obviously brighter in (a), while nearly no fluorescent signal on the surface of negative control microspheres in (b), which was well consistent with our expectation. The sizes of QD-probes affect the labeling efficiency to the microspheres due to the steric hindrance [27]. The big-sized QD-probes had lesser capacity of binding onto the surface of PS microspheres than that of small-sized ones. The QD-probes in the present experim ent were smaller than 10 nm. Thus, the fluorescence signal on the positive control microspheres was homogeneous and delicate. Non-spe cific adsorption should be avoided and eliminated in immuno fluorescence detection. The water-solubilization of hydrophobic QDs can eliminate non-specific adsorption to the microspheres. The as-prepared water-soluble QDs in this paper, with abundant surface carboxyl groups on their surfaces, had good water-soluble
Normalized PL intensity
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ability. No precipitation had been found even if they were put in a refrigerator at 4 °C for a long time. The QDs–IgG bioconjugates had better water-solubility and lower charge density since IgG mole cules had attached to the QDs, which could further reduce nonspecific adsorption. In order to test the sensitivity of quantum dots/particle-based immunofluor escence assay, different concentrations of AFP anti gen were added to the microspheres coated with mouse anti-AFP McAb. Fluorescent intensities were measured after the correspond ing QDs-mouse anti-AFP McAb bioconjugates added. The diagnos tic scheme is shown in Scheme 1. Fig. 4 shows the fluorescence emission spectra of the sandwich immunoassay. It can be seen that the fluorescent intensity of QDs labeled mouse anti-AFP McAb at 612 nm increased gradually with the increasing concentration of AFP antigen added. By mathematical analysis, the detection limit of AFP antigen is 4.9 ng/mL. For further tapping the potential of particle-based immuno assay, superparamagnetic particle-based immunofluorescence assay was constructed. Magnetic PS microspheres were prepared by doping hydrophobic ferric oxide into the matrix of PS micro spheres. Magnetic particles permit fast and easy separation of solid and liquid phases [28]. Magnetic measurements on hydrophobic ferric oxide nanocrystals and magnetic PS microspheres shown in
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Fig. 3. Fluorescence/optical microscopy photographs, the immunofluorescence images of human IgG sensitized PS microspheres treated with QD-anti-human IgG probes; (a) positive control, QD-anti-mouse IgG probes; (b) negative control; the optical microscopy photograph of magnetic microspheres; (c) the immunofluorescence images of human IgG sensitized magnetic PS microspheres treated with QD-anti-human IgG probes; (d) positive control, QD-anti-mouse IgG probes and (e) negative control.
Fig. 5 indicated that the particles were superparamagnetic at room temperat ure. Under a large external field, the magnetization of the particles aligned with the field direction and reached its saturation value (saturation magnetization). The saturation magnetization values of hydrophobic ferric oxide nanocrystals and magnetic PS microspheres were 67.68 and 26.43 emu/g, respectively at 300 K.
And the magnetic remanences of the samples were nearly zero with S-like curves in shape, which indicated the synthesized particles exhibited superparamagnetism. The surface morphol ogy of PS microspheres after doping in the organic solution were observed using microtechnique as shown in Fig. 3c. Rough surface easily results in an increase of non-specific adsorption [29], which
Scheme 1. Diagnostic scheme. (A) Diagram illustrating the integration of PS microspheres, magnetic nanoparticles and QDs, with solution-based sandwich assay for AFP fluorescence detection. (B) Diagram illustrating the magnetic separation under an external magnet.
B. Zhang et al. / Journal of Photochemistry and Photobiology B: Biology 94 (2009) 45–50
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ferric oxide into the matrix of PS microspheres. Various character izations showed that the as-prepared water-soluble QDs and mag netic microspheres were well defined. Immune QD-probes and PS microspheres were prepared via covalent attachment of water-sol uble QDs and carboxyl-PS microspheres to IgG. The distinguishable immunoreactions happened on the PS microspheres indicated that the QDs would be another smart assistant for the particle-based immunoassays. Furthermore, the detection sensitivity of current assay is tested with sandwich immunofluorescence assay using AFP as antigen model. The method described in this paper could provide a reference and strategy for application of QDs in particlebased immunoassays.
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This work was supported by the 863 Program (No. 2007AA021808) and National Science and Technology Program for Key Projects (No. 2004BA519A56-04). References
should be avoided in this experiment. Fortunately, the micro spheres kept spherical with smooth surface, since the cross-linked PS microspheres had resistance to the organic solvent. Human IgG coated magnetic PS microspheres were obtained with the same procedure for the blank PS microspheres. The contrastive fluores cent images as shown in Fig. 3d/e also indicated that the binding of QD-probes to the antigen sensitized magnetic PS microspheres was specific and responsible. Magnetic PS microspheres are used as solid support for immunoreactions have some advantages, such as easy separation under an external magnetic field and biomole cules concentration. 4. Conclusion Immunofluorescence assay based on QD-probes and PS micro spheres was constructed in this paper. High quality water-soluble CdSe/ZnS QDs were synthesized based on the self-assembling on the surface of hydrophobic CdSe/ZnS QDs using amphiphilic poly mer. Magnetic microspheres were obtained by doping hydrophobic
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Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / j p h o t o b i o l
Quantum dots/particle-based immunofluorescence assay: Synthesis, characterization and application Bingbo Zhang a, Xiaofei Liang a, Lijuan Hao a, Jing Cheng a, Xiaoqun Gong a, Xuhui Liu b, Guiping Ma b, Jin Chang a,* a b
Institute of Nanobiotechnology, School of Materials Science and Engineering, Tianjin University, No. 92 Weijin Road, Nankai District, Tianjin 300072, China Center of Technology, Beijing Entr-exit Inspection and Quarantine Bureau, Beijing 100029, China
a r t i c l e
i n f o
Article history: Received 2 May 2008 Received in revised form 14 September 2008 Accepted 29 September 2008 Available online xxxx Keywords: Quantum dots Polystyrene microspheres Magnetic Fluorescent Immunofluorescence assays
a b s t r a c t Recently, it has been proved that quantum dots (QDs) hold the potential to be used in the bioanalysis as fluorescent probes for their many unique optical properties. In this paper, immunofluorescence assay, an integration of particle-based immunoassays and fluorescent QD-probes, was constructed. Firstly, high qual ity CdSe/ZnS QDs were prepared. Then after being water-solubilized by amphiphilic polymer based on selfassembling, the QDs were labeled by immunoglobulin G (IgG) antibody. At the same time, both carboxylpolystyrene (PS) and magnetic carboxyl-PS microspheres were prepared and coated by antigens. The antigen sensitized PS microspheres were specifically captured by the QD–IgG bioconjugates based on the antibodyantigen reaction, which was confirmed by the immunofluorescence test in vitro. The sensitivity of current assay was tested by sandwich immunofluorescence assay using human alpha fetoprotein (AFP) as antigen model. The detection limit of AFP antigen is 4.9 ng/mL. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Particle capture enzyme-linked immunosorbent assay (ELISA) tests, based on the very specific interaction of antibody and anti gen, have been in common use, since latex particles or micro spheres were first used in medical diagnostic application as latex agglutination test (LAT) in the late 1950s [1]. Nowadays, there have been many innovative improvements based on the latex particles including colored particles [2], special devices for results inter pretation [3–5], magnetic particles [6], and integration of parti cles and fluorescent report probes [4,7]. Magnetic particles can be used to help pull things out of solution more quickly, which offers an easy solid–liquid separ ation [8]. Integrated with report probes, especially fluorescent probes, particles-based immunoassays have attracted great attentions from biologist, chemist, and materials researchers. The fluorescent probes used in immunofluorescence assays are usually conventional fluorophores/dye [7]. Those fluoro phores are not suitable for extended periods of observations using fluorescent and confocal microscopy because organic fluorophores tend to quench rapidly [9]. Furthermore, it is sometimes dif ficult or impossible to record fine fluorescent images while the organic dye probes fade in the course of adjusting the focus. In contrast, QDs, also called inorganic nanocrystals [10–14], are stabilized over a far longer exposure-time to light and can emit a fluorescence of high * Corresponding author. Tel./fax: +86 22 27401821. E-mail address: jin[email protected] (J. Chang). 1011-1344/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2008.09.008
luminosity at an almost equivalent condition as the conventional organic dye probes. Besides that, QDs have many other advanta ges over traditional organic dyes: tunable fluorescent wavelength by raw materials and size, broad excitation spectra, sharp and symmetrical fluorescent peak, large Stokes shift, and differentsized QDs can be excited simultaneously by a single wavelength [15]. Problems with organic fluorophores include narrow excita tion bands and broad emission spectra, which makes detection of multiple color emitting probes dif ficult due to spectral over lap. With improvement of preparation of water-soluble QDs, they have shown increasing promising applications in biological fields, especially as fluorescent probes for biological markers, biological detection and biological imaging area [16–18]. In this paper, we described a novel method, integration of particles and QDs, for preliminary biodetection. Firstly the carboxyl-PS and magnetic carboxyl-PS microspheres were covalently attached by antigen/ antibody. Then the sensitized microspheres acted to magnify the antibody-antigen reaction when they were mixed with antibodylabeled QD-probes. 2. Materials and methods 2.1. Materials and apparatus Selenium powder (100 mesh, 99.99%, Aldrich), Cadmium oxide (CdO, 99.5%, Aldrich), Zinc oxide (ZnO, 99.9%, Sigma), Sulphur (99.98%, Aldrich), Tri-n-butylp hosp hine (TBP, 90%,TCI, Japan),
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B. Zhang et al. / Journal of Photochemistry and Photobiology B: Biology 94 (2009) 45–50
Tri-n-octylphosphine oxide (TOPO, 90%, Aldrich), Octadecylamine (ODA, 90%, ACROS), 1-Octadecene (ODE, 90%, ACROS), Oleic acid (OA, 90%, Aldrich), Octylamine (99%, Aldrich). Carboxyl- PS micro spheres (Tianjin BaseLine ChromTech Research Centre, 7–8 lm), 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC, GL Biochem (Shanghai) Ltd.), Human IgG: purified total IgG from normal human serum, in which heavy chain is 50.000 Da, light chain is 25.000 Da. Goat anti-human IgG: pure human total IgG immune against goat until 1:100000 (ELISA), Goat anti-mouse IgG: pure mouse total IgG immune against goat until 1:100000 (ELISA),above supplied by BEIJING DINGGUO BIOTECHNOLOGY CO.LTD. Human alpha fetoprotein (AFP) antigen, mouse monoclo nal antibody to human AFP (Clone: A3 for PS microspheres coat ing, Clone: D2 for QDs labeling), supplied by Beijing Keyuebio BiotechCo. Ltd. chloroform, ethanol, dichloromethane and argon (oxygen free) were obtained from local suppliers. All chemicals were used without further purification. Ultraviolet–visible (UV–vis) absorption spectra were recorded on UV-2450 spectrophotometer. Photoluminescence (PL) mea surement was performed at room temperat ure using an F-4500 (HITACHI) spectrophotometer. QDs and hydrophobic ferric oxide nanocrystals were visualized using a Tecnai G2 F20 transmission electron microscope (TEM) operating at an accelerat ion voltage of 200 kV. The magnetic property of PS microspheres was analyzed at Vibrating Samples Magetometer (VSM), LDJ9600-1. Immu nofluor escence assay were examined under an OLYMPUS.BX51 fluorescence microscope equipped with an OLYMPUS MICRO DP 70 camera. 2.2. Synthesis of water-soluble amphiphilic triblock copolymer-capped CdSe/ZnS QDs CdSe/ZnS QDs were synthesized according to the previously reported procedure [19]. However, amphiphilic triblock copolymer was selected as the surface capping reagent to form water-soluble QDs for labeling IgG. Triblock copolymer from Sigma consists of a polybutylacrylate segment, a polyethylacrylate segment and a poly methacrylic acid segment. At a molecular weight of ca.100,000 Da, for better encapsulation of QDs, about 25% of the free carboxylic acid groups were derivatized with octylamine (a hydrophobic side chain). CdSe/ZnS QDs and amphiphilic triblock polymer (The mol ratio of QDs to polymer was 1:10) were dissolved in chloroform/ ethanol solvent mixture (3:1, v/v) and stirred for several hours for chloroform evaporation. After evapor ation of chloroform, the encapsulated dots were suspended in PBS, sonicated for 3 min and then purified by gel filtration (sepharose 6 fast flow, GE healthcare life sciences).
as coupling agent in the PBS buffer for 2.0 h. The mol ratios ofQDs/ IgG/EDC were in 1:15:4000. The final QDs–IgG bioconjugates were purified by ultracentrifugation, and then dissolved in the PBS (0.01 M, pH 7.2, 0.5%BSA). Highly carboxylated PS microspheres, including ferric oxide doped carboxyl-PS microspheres, were both coated with human IgG via the covalent bonds between human IgG molecules and microspheres. The solutions of human IgG (1.0 mg/mL, 0.1 mL), EDC (10 mg) and microspheres (10 mg/mL, 1.0 mL) were gently mixed in the PBS buffer for two hours at room temperature using an orbital shaker followed by storage at 4 °C. The above coupling protocols are feas ib le for the micros pheres coati ng with mouse anti-AFP McAb, and QDs labeling with mouse anti-AFP McAb. 2.5. Immunofluorescence assay The immunofluorescence assay for the positive and negative control experiments were both carried out in the same manner. QD-goat anti-human IgG bioconjugates were used as the positive control, while QD-goat anti-mouse IgG bioconjugates as the neg ative control. The human IgG-coated PS microspheres (2.0 mg/mL, 1.0 mL) were firstly blocked for 30 min at room temperature in the PBS–BSA buffer (0.01 M, pH 7.2, 0.5%BSA), then gently mixed with QD-goat anti-human IgG bioconjugates (16 nmol/L, 0.1 mL) and QD-goat anti-mouse IgG bioconjugates (16 nmol/L, 0.1 mL) sep arately for 45 min at room temperature, using an orbital shaker. After antibody-antigen reaction, centrifugations/PBS-T (0.01 M, pH 7.2, 0.05% Tween) redispersion procedures were applied to separate the unbound QD–IgG bioconjugates, since the PS micro spheres can be centrifugated down at 4000 rpm/1 min, while unboundQD–IgG bioconjugates still suspended. Finally the micro spheres were examined under an OLYMPUS.BX51 fluorescence microscope equipped with an OLYMPUS MICRO DP 70 camera and a broad band light source (ultraviolet 330–385 nm). The illumi nation light was from an O-LH100HG 100 W mercury lamp with Automatic exposure control. The sandwich assay was used to test the detection limit of AFP antigen. The mouse anti-AFP McAb (Clone: A3) coated PS micro spheres and different concentration of AFP antigen were mixed and incubated on a shaker for 1 hour, and then wash three times to remove the unbound antigens. The PS microspheres/AFP antigen complexes were obtained. Then QDs-mouse anti-AFP McAb(Clone: D2) bioconjugates were added. The reaction system was incubated with gentle shaking for another 1 h and followed by centrifuga tion. The sediments were resuspended in PBS buffer for following study. 3. Results and discussion
2.3. Preparation of ferric oxide doped carboxyl-PS microspheres Hydrophobic ferric oxide was synthesized according to a pre viously reported protocol with minor modifications [20]. Embed ding of PS beads was accomplished by doping a certain amount of ferric oxide into carboxyl-PS microspheres. Firstly the micro spheres (5.0 mg) were swelled in a solvent mixture containing chloroform and butanol (5:95, v/v) to improve the capability of absorbing ferric oxide in a centrifugal tube. Then 0.1 mg of ferric oxide dissolved in chloroform solvent was added into the tube and stirred for 2–4 h until the ferric oxide penetrate into microspheres adequately. Finally, the microspheres were washed with ethanol. 2.4. Water-soluble QDs and carboxyl-PS microspheres labeling with IgGs The as-prepared water-soluble QDs were reacted with goat anti-human IgG and goat anti-mouse IgG, respectively using EDC
The dispersion and particle sizes of polymer modified CdSe/ZnS QDs and hydrophobic ferric oxide nanocrystals were evaluated by a transmission electron microscopy (TEM) as shown in Fig. 1. The particles of polymer modified QDs were well dispersive without obvious aggregation due to electrostatic repulsion from the car boxylic groups on the surface of each QD [21]. TEM analysis also showed the prepared hydrophobic ferric oxide nanocrystals were monodisperse. Fig. 2a was the UV–visible absorption spectra of the polymer modified CdSe/ZnS QDs. The spectra showed that QDs had a broad and continuous absorption, allowing excitation by a wide range of wavelengths. And it kept the original optical property after water solubilization. Fluorescence emission spectra in Fig. 2b showed peaks both narrow and symmetric with no tailings. The red-shift of peak from 604 nm to 611 nm after water solubilization proce dure indicates that the surface environment of CdSe/ZnS changed from oil phase to water phase [22,23]. And the full width at half
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Fig. 1. TEM image of polymer modified CdSe/ZnS QDs.
axim m um (FWHM) of fluorescence emission peaks broaden slightly from 27 nm to 29 nm. Planar assays based on flat carriers (glass sides, flat-bottomed ELISA plate, et al.) are among the most intensively investigated for bioassays [24]. However, suspension array based on particles is more promising since its inherent and unique characteristics. The high binding capacity of three-dimensional microspheres makes the suspension array a very sensitive platform for immunofluores cence assays. One of the most used carriers in suspension array is particles or beads. The encoded particles have great potentials for bioapplication, for example, being integrated with a flow cyto metric system [25], or Luminex flow analyzer [26] to make a highthroughput screening. Carboxyl-PS microspheres, which were easily recognizable with the optical microscopy, were used in this paper to be the solid supports for immobility of antigen via the covalent attachment. Human IgG were tagged onto the surface of microspheres using EDC as coupling agents for both positive and negative control experiments. Herein, fluorescent report QDprobes were used instead of the traditional dye molecule-probes. The QD-anti-human IgG bioconjugates were used as the positive probes, while the QD-anti-mouse IgG bioconjugates were used as the negative probes. Fig. 3a/b shows the immunofluorescence images of human IgG sensitized PS microspheres treated with QDprobes. The images showed that the fluorescence on the positive control microspheres was obviously brighter in (a), while nearly no fluorescent signal on the surface of negative control microspheres in (b), which was well consistent with our expectation. The sizes of QD-probes affect the labeling efficiency to the microspheres due to the steric hindrance [27]. The big-sized QD-probes had lesser capacity of binding onto the surface of PS microspheres than that of small-sized ones. The QD-probes in the present experim ent were smaller than 10 nm. Thus, the fluorescence signal on the positive control microspheres was homogeneous and delicate. Non-spe cific adsorption should be avoided and eliminated in immuno fluorescence detection. The water-solubilization of hydrophobic QDs can eliminate non-specific adsorption to the microspheres. The as-prepared water-soluble QDs in this paper, with abundant surface carboxyl groups on their surfaces, had good water-soluble
Normalized PL intensity
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ability. No precipitation had been found even if they were put in a refrigerator at 4 °C for a long time. The QDs–IgG bioconjugates had better water-solubility and lower charge density since IgG mole cules had attached to the QDs, which could further reduce nonspecific adsorption. In order to test the sensitivity of quantum dots/particle-based immunofluor escence assay, different concentrations of AFP anti gen were added to the microspheres coated with mouse anti-AFP McAb. Fluorescent intensities were measured after the correspond ing QDs-mouse anti-AFP McAb bioconjugates added. The diagnos tic scheme is shown in Scheme 1. Fig. 4 shows the fluorescence emission spectra of the sandwich immunoassay. It can be seen that the fluorescent intensity of QDs labeled mouse anti-AFP McAb at 612 nm increased gradually with the increasing concentration of AFP antigen added. By mathematical analysis, the detection limit of AFP antigen is 4.9 ng/mL. For further tapping the potential of particle-based immuno assay, superparamagnetic particle-based immunofluorescence assay was constructed. Magnetic PS microspheres were prepared by doping hydrophobic ferric oxide into the matrix of PS micro spheres. Magnetic particles permit fast and easy separation of solid and liquid phases [28]. Magnetic measurements on hydrophobic ferric oxide nanocrystals and magnetic PS microspheres shown in
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Fig. 3. Fluorescence/optical microscopy photographs, the immunofluorescence images of human IgG sensitized PS microspheres treated with QD-anti-human IgG probes; (a) positive control, QD-anti-mouse IgG probes; (b) negative control; the optical microscopy photograph of magnetic microspheres; (c) the immunofluorescence images of human IgG sensitized magnetic PS microspheres treated with QD-anti-human IgG probes; (d) positive control, QD-anti-mouse IgG probes and (e) negative control.
Fig. 5 indicated that the particles were superparamagnetic at room temperat ure. Under a large external field, the magnetization of the particles aligned with the field direction and reached its saturation value (saturation magnetization). The saturation magnetization values of hydrophobic ferric oxide nanocrystals and magnetic PS microspheres were 67.68 and 26.43 emu/g, respectively at 300 K.
And the magnetic remanences of the samples were nearly zero with S-like curves in shape, which indicated the synthesized particles exhibited superparamagnetism. The surface morphol ogy of PS microspheres after doping in the organic solution were observed using microtechnique as shown in Fig. 3c. Rough surface easily results in an increase of non-specific adsorption [29], which
Scheme 1. Diagnostic scheme. (A) Diagram illustrating the integration of PS microspheres, magnetic nanoparticles and QDs, with solution-based sandwich assay for AFP fluorescence detection. (B) Diagram illustrating the magnetic separation under an external magnet.
B. Zhang et al. / Journal of Photochemistry and Photobiology B: Biology 94 (2009) 45–50
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ferric oxide into the matrix of PS microspheres. Various character izations showed that the as-prepared water-soluble QDs and mag netic microspheres were well defined. Immune QD-probes and PS microspheres were prepared via covalent attachment of water-sol uble QDs and carboxyl-PS microspheres to IgG. The distinguishable immunoreactions happened on the PS microspheres indicated that the QDs would be another smart assistant for the particle-based immunoassays. Furthermore, the detection sensitivity of current assay is tested with sandwich immunofluorescence assay using AFP as antigen model. The method described in this paper could provide a reference and strategy for application of QDs in particlebased immunoassays.
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This work was supported by the 863 Program (No. 2007AA021808) and National Science and Technology Program for Key Projects (No. 2004BA519A56-04). References
should be avoided in this experiment. Fortunately, the micro spheres kept spherical with smooth surface, since the cross-linked PS microspheres had resistance to the organic solvent. Human IgG coated magnetic PS microspheres were obtained with the same procedure for the blank PS microspheres. The contrastive fluores cent images as shown in Fig. 3d/e also indicated that the binding of QD-probes to the antigen sensitized magnetic PS microspheres was specific and responsible. Magnetic PS microspheres are used as solid support for immunoreactions have some advantages, such as easy separation under an external magnetic field and biomole cules concentration. 4. Conclusion Immunofluorescence assay based on QD-probes and PS micro spheres was constructed in this paper. High quality water-soluble CdSe/ZnS QDs were synthesized based on the self-assembling on the surface of hydrophobic CdSe/ZnS QDs using amphiphilic poly mer. Magnetic microspheres were obtained by doping hydrophobic
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