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Synthesis and Characterization of Double-layer Quantum-Dots-Tagged Microspheres Xinghua Pan, Maolin Lu, Daocheng Wu∗ , and Lili Gai
Abstract—Quantum - dots - tagged poly ( styrene - acrylamide acrylic acid) microspheres (QDsAAMs) were synthesized and modified with hydrazine hydrate through hydrazinolysis. Azidocarbonyl groups, which can be rapidly coupled with proteins under mild conditions, were introduced onto the surface of QDsAAM using azido reaction. Bovine serum albumin (BSA) was selected as model protein to be covalently immobilized on the azidocarbonyl QDsAAM. Instruments such as fluorescence microscope, optical microscope, confocal laser scanning microscope, UV–visible spectrometer, Fourier transform infrared spectrometer, size analyzer, and fluorescence spectrophotometer were used to characterize QDsAAM. Results showed that QDsAAM had a regular double-layer spherical shape and an average diameter of 11.2 µm. It also displayed high fluorescence intensity (λex /λem = 250 nm/ 370 nm), which showed linearity with concentrations ranging from 3.0 × 10−3 to 90.0 × 10−3 g·L−1 . In addition, external factors such as pH and ionic strength exerted little influence on fluorescent characteristic. BSA immobilization indicated that QDsAAM with azidocarbonyl groups could be covalently coupled with BSA at the rate of 40 × 10−3 g/g (BSA/QDsAAM), while fluorescence linearity correlation was also found. This functional azidocarbonyl QDsAAM with sensitive fluorescence and active azidocarbonyl groups could be used as a promising fluorescent probe for quantitative detection, protein immobilization, and early rapid clinical diagnostics. Index Terms—Azidocarbonyl groups, double layer, fluorescence characteristics, quantum dots (QDs) microspheres.
I. INTRODUCTION ECENTLY, functional polymer microspheres have been widely used in biomedical fields such as bioseparation, enzyme immobilization, and controlled release [1]–[4]. Their widespread application is due to their unique characteristics that include possession of a large specific surface area, stability, and capability to undergo coupling with biological molecules. Among various microspheres, fluorescent microspheres have attracted great attention in various fields, such as labeling [5], [6], immunoassay study [7], and flew cytometric analysis [8], [9]. These fluorescent microspheres possess high brightness and im-
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Manuscript received April 30, 2008. First published March 16, 2009; current version published June 24, 2009. This work was supported by the National Natural Science Foundations of China under Grant 30772658 and Grant 30570494. Asterisk indicates corresponding author. X. Pan, M. Lu, and L. Gai are with the Key Laboratory of Biomedical Information Engineering of the Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China. ∗ D. Wu is with the Key Laboratory of Biomedical Information Engineering of the Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China (e-mail:
[email protected]. edu.cn). Color versions of one or more of the figures in this paper are available online at http://ieeexplore.ieee.org. Digital Object Identifier 10.1109/TNB.2009.2016549
proved photostability compared with conventional fluorescent dyes; this is because they not only offer sensitive detection by the analysis of their fluorescent properties but they can also be more flexible in immunolabeling [10]. As comparatively new colloidal nanocrystals semiconductors, quantum dots (QDs) have likewise attracted increasing attention due to their special characteristics. Compared with organic dyes, QDs show tunable fluorescence signatures, narrow emission spectra, brighter emission, and good photostability [11], [12]. However, they can easily aggregate in acidic and isotonic conditions once they become unstable. Hence, it is difficult to produce QDs-conjugated biomolecules because most of them exist in the isotonic condition in vivo [13]. At present, one of the methods used to overcome this obstacle is incorporating QDs into polymer microspheres, which can stably exist in both acidic and isotonic conditions. However, potential deficiencies such as the difficulty of encapsulating QDs in polymer microspheres due to poor aqueous-phase transportation prevent their effective application in practice. Additionally, although the synthesis of different kinds of polymer-bearing QDs has become a hotspot [14]–[17], when considering pragmatic problems of bioconjugation with biomolecules and QDs leakage, most available QDs microspheres are either lacking in functional groups or require a laborious process of group fabrication. Hence, it is desirable to prepare QDs-tagged microspheres with facile functional groups, which could be conveniently and efficiently coupled with biomolecules, and can also effectively prevent QDs from leaking. At the same time, based on our knowledge, few reports have publicly referred to a quantitive determination of QDs microspheres with linearity correlation between fluorescence intensity and concentrations of fluorescent microspheres. In this paper, we attempt to develop functional QDs-tagged microspheres with active surface functional groups, which may offer advantages due to their ability to easily combine with biomacromolecules, their inherent sensitive fluorescent characteristics, and the minimal amount of QDs leakage resulting from their use. According to this strategy, QDs-tagged poly(styreneacrylamide-acrylic acid) microspheres (QDsAAM) were synthesized. Surface modifications were performed that consist of hydrazinolysis of the amide group to the hydrazide group and diazotizing of the hydrazide group to the azidocarbonyl group. Chemical coupling between bovine serum albumin (BSA) and QDsAAM took place through a displacement reaction of the protein’s amino groups onto the azidocarbonyl groups on QDsAAM. The reaction sequence is illustrated in Fig. 1, and the processes are simple, rapid, and easy to control. Moreover, the cumulative results of fluorescence measurements, Fourier
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Fig. 1. Schematic illustration of fabrications of QDsAAM, hydrazide QDsAAM, protein-immobilized QDsAAM.
transform infrared spectrometer (FTIR) spectra, and the high entrapment efficiency of QDs in the QDsAAM indicate that QDs were embedded into the microspheres and that QDsAAM possessed superior fluorescent characteristics. Experiments show that the amount of BSA covalently immobilized is 40 × 10−3 g/g (BSA/QDsAAM). Therefore, the application of QDsAAM with surface azidocarbonyl groups is a promising method that can provide a new reagent for early and rapid clinical diagnostics, as well as immunological studies in the future. II. EXPERIMENTAL METHOD A. Material Styrene (chemically pure, Chinese Medical Chemicals Company Ltd., Shanghai, China) was distilled under reduced pressure to remove the inhibitor. Other chemicals were acrylamide (analytical reagent, Amresco Fraction, USA), acrylic acid (analytical reagent, Third Chemical Plant, Tianjin, China), and potassium persulfate (KPS, analytical reagent, Chemical Plant, Xi’an, China). Core/shell CdSe/ZnS QDs were kindly provided by Prof. D. Pang (Wuhan University, China). The core/shell CdSe/ZnS QDs were prepared by covering the core CdSe nanocrystals with a thin but higher bandgap material ZnS to yield QDs with strong light emission and high photostability, and aminoacetic acid was modified onto the surface of CdSe/ZnS [19]. BSA (Mr 68000, Roche Fraction, Germany) of analytical grade was obtained commercially and used without further purification. B. Synthesis 1) Synthesis of QDsAAM: QDsAAM was prepared by modified soapless emulsion polymerization that was carried out in a three-necked separator flask equipped with a stirrer, spiral condenser, and dropping funnel with an inlet. Double distilled water was boiled first to remove oxygen. In the first step, 4 mL of QDs (8 µmol·L−1 ), 0.1 g KPS, and 0.5 mL of sodium dodecyl sulfate (SDS, 1%) were dispersed in 10 mL of ethanol solution. The obtained ethanol phase mixture was sonicated for
10 min by an ultrasonic cleaner (KS-3200DE, Kunshan, Jiangsu, China) and was kept at room temperature for 20 h. Afterwards, 0.5 g acrylamide was added into the three-necked separator flask with 18.3 mL of double distilled water while stirring. After this, 3.6 mL of styrene and 3.3 mL of ethanol were mixed with the ethanol phase containing QDs. The mixture was added dropwise in the aqueous phase to be dispersed for 20 min. In the second step, 0.066 g KPS and 0.1 g NaCl were added into the reactor to initiate the polymerization process when the time was recorded at zero point of the reaction. Five minutes later, the addition of 0.16 g acrylic acid was followed by pH adjustment with 1 mol·L−1 NaOH. The polymerization was allowed to proceed at 70 ◦ C in still N2 atmosphere for 10 h. Subsequently, another 0.016 g of KPS and 0.032 g of NaCl were added into the system. The system was then cooled to 4 ◦ C, and the polymerization was allowed to continue for another 9 h. Afterwards, the production was purified three times by dialysis carried out with distilled water. Finally, it was lyophilized to primrose pink powder and stored at 4 ◦ C prior to use. 2) Synthesis of Hydrazide QDsAAM: The fabrication of the surface functional groups on the homogeneous QDsAAM, as described in this section, was done analogous to the described method [20]. It also comprised a two-step reaction sequence. In the first step, the QDs fluorescent microspheres bearing the hydrazide groups on the surface, known as hydrazide QDsAAM, were fabricated by hydrazinolysis. Briefly, 20 mL of QDsAAM (5%) was added into a flask under continuous magnetic stirring. The hydrazide reaction was allowed to proceed at 45 ◦ C for 8 h, followed by the addition of excess hydrazine hydrate (80%). After being left to cool at room temperature, the product was purified by dialyzing hydrazide QDsAAM for several times, using distilled water, which can remove unreacted hydrazine until the pH value was 7.0. It was then stored at 4 ◦ C prior to use. In the second step, the functional azidocarbonyl QDs fluorescent microspheres (azidocarbonyl QDsAAM) were prepared by azido reaction. Briefly, the pH of a certain volume of hydrazide QDsAAM suspension (4%, 5.0 mL) was adjusted to 1.0–2.0, with 1.0 mol·L−1 HCl solution under magnetic stirring. Afterwards, 0.1 mol·L−1 NaNO2 solution was added dropwise into the suspension until the color of the starch KI indicator paper changed within a period of about 1 min. The suspension was stirred at 0 ◦ C–5 ◦ C for 1 h. Further purification was carried out at 4 ◦ C. 3) Synthesis of Protein-Immobilized QDsAAM: BSA was selected as model protein to test the immobilization characteristic of the functional azidocarbonyl QDsAAM. Initially, 2.5 mL of freshly azidocarbonyl QDsAAM (4%) was added into a reactor. The pH was adjusted to 8.0 with 1.0 mol·L−1 NaOH solution at 0 ◦ C–5 ◦ C under magnetic stirring. BSA (230 µL, 5%, pH 8.0) was diluted by slowly adding the PBS buffer solution (pH 8.0, 0.01 mol·L−1 ) into the reactor. Afterwards, the system was stirred at 0 ◦ C–5 ◦ C for 5 h. Glycin (500 µL, 2.5%, pH 8.0) was added to discharge the unreacted azidocarbonyl groups, and then the mixture was dialyzed by double distilled water to wash away salt, ion, and other small molecular materials. Finally, the mixture was separated through Sephadex G-25 media column,
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and the supernatants were collected for an analysis of BSA concentration. Some measures were taken to minimize the loss of BSA such as the repeated wash of the dialysis membrane. C. Particle Size and Morphology of QDsAAM An optical microscope (OLYMPUS CX 41, Japan) was used to examine the morphology of QDsAAM. To verify the results obtained through the optical microscope, the average diameter of QDsAAM were measured by size analyzer (Malvern mastersizer 2000, Malvern instruments Ltd., U.K.). At the same time, a confocal laser scanning microscope (Leica Microsystems Heidelberg GmbH, Germany) was used to detect the location of QDs inside QDsAAM. D. Functional Groups and Immobilization of Protein Using KBr pellets, the functional groups on the surface of QDsAAM were evaluated by a FTIR (IRpresitge-21, Japan). In contrast, the FTIR spectra of QDs and poly(styrene-acrylamideacrylic acid) microspheres without QDs were also recorded. The immobilization capacity of BSA was determined by UV spectrophotometer (ultrospec 2100pro, Amersham Biosciences, Sweden), while the unbound BSA collected through the column separation was measured according to a calibration curve. The amount of BSA immobilized on the functional azidocarbonyl QDsAAM was then determined by measuring the initial and unbound BSA concentrations. The experiment was repeated for three times. E. Fluorescent Characteristics The fluorescence of QDs solution, QDsAAM, hydrazide QDsAAM, poly(styrene-acrylamide-acrylic acid), and proteinimmobilized QDsAAM aqueous solutions with pH 7.0 were all estimated using a fluorescence spectrophotometer (HORIBA Jobin Yvonn, FluoroMax-4, USA) at room temperature. At the same time, a fluorescence microscope (OLYMPUS CX41, Japan) was used to facilitate intuitive fluorescence examination of QDsAAM. III. RESULTS AND DISCUSSION A. Preparation of QDsAAM In general, the preparation of QDs composite microspheres needs to address the crucial problem of incorporating the QDs into the polymer matrix. Two common methods, encapsulation and adsorption, were used to perform this process. The adsorption process was generally based on the porous structure of the support matrix or hydrogel as the adsorbed material in the outer layer tends to leak out if no outside covering is placed. On the other hand, the encapsulation process involves mixing the monomer and the encapsulated material before the polymerization starts. This could make the encapsulated material disperse homogeneously in the monomer media, although it might have an effect on the kinetics and mechanism of polymerization. During the polymerization process, it is critical to ensure that monomer polymerization occurs near the surface of the QDs,
Fig. 2.
Optical microscopy image of QDsAAM.
i.e., the nucleation regions. It is also necessary to maintain the stability of the growing composite particles so as to deaden both coalescence and the further breakup of microspheres caused by the interaction of QDs. Unfortunately, in conventional emulsion polymerization, it is difficult to obtain the QDs composite microspheres because nucleation regions are plagued by micelles. Therefore, the stability of the system is weakened in the presence of QDs acting as strong inhibitors to the polymerization process. Currently, two modification measures were adopted in this paper. First, a 0.5 mL of SDS solution (1%, less than its CMC 10.0 × 10−3 mol·L−1 ) was added into the polymerization system as an emulsifier to prevent the formation of micelles and to ensure the stability of the QDs composite particles. Second, the QDs were directly modified by aminoacetic acid to obtain amphiphilic structure for inducing the initialization process to occur near the surface of QDs. Although the pretreatment steps described previously were performed, QDs could still be absorbed on the polymer surface, which might lead to a leakage problem during application. To address this problem in this study, a double-layer structure was fabricated to eliminate the surface adsorption as much as possible. The initiator was added at two different points to initialize the reaction in an appropriate pace instead of adding a large amount at a single point. Subsequently, at the beginning of the second initialization, temperature was decreased significantly to slow down the reactive velocity. As the temperature fell, the energy of molecular motion decreased, leading to the mild fabrication rate of the outer layer. The experiment results suggested that this method for preparing QDsAAM was feasible. Moreover, it is also suitable for massive production as it uses simple procedures and a double-layer structure, which can easily be manipulated. Its encapsulation ratio is over 80% according to the initial QDs concentration. B. Morphology and Particle Size of QDsAAM We can see the double-layer structure from Fig. 2, with the inner layer having an irregular shape whereas the outer layer
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Fig. 5. FTIR spectra of (a) poly(styrene-acrylamide-acrylic acid) microspheres, (b) QDs, and (c) QDsAAM.
was no significant change in the morphology and particle size of QDsAAM, demonstrating stability and good fluidity. C. Functional Groups
Fig. 3. (a) Fluorescent microscopy image of QDsAAM. (b) Confocal laser microscopy sections image of QDsAAM.
Fig. 4.
Size distribution of QDsAAM.
exhibits smooth sphericity. As shown in Fig. 3(a), QDsAAM can give off fluorescence when excitated, and the prepared QDsAAM have a regular spherical shape that coincides with Fig. 2. At the same time, from the series scanning pictures in Fig. 3(b), we can see two different QDs distributions inside microspheres. The inner layer that seems irregular has concentrated QDs, while other parts take only a little of the QDs. This confirmed the successful embedment of QDs into the polymers and double-layer structure of the microspheres. On the other hand, Fig. 4 indicates that 75.2% of the QDsAAM have a diameter from 5.03 to 19.9 µm, with an average of 11.2 µm. In addition, after being stored under common conditions such as in a refrigerator or at room temperature for six months, there
In practical applications, the immobilization of proteins and other macromolecules can be performed using several methods such as adsorption, entrapment, and chemical binding. The major advantage of the chemical binding method is that it can provide a steady and certain linking site to the target as compared to adsorption and the entrapment method. However, covalent binding requires that the supports should have suitable groups on the surface. Many groups such as hydroxyl, amine, carboxyl, and aldehyde groups have been introduced onto the surface for chemical binding or for further modification. Comparatively, the amino groups have very high stability and reactivity, and can also covalently link to the protein in mild conditions after two-step modification. As compared to surface modification, copolymerization was proven to be an easier method when the groups were introduced onto the surface of the polymer. In our study, acrylamide and acrylic acid were selected as the functional monomers and were included in the copolymerization process, thereby resulting in the simultaneous completion of the surface functionalized procedure. The FTIR spectra illustrated in Fig. 5 include embracing poly(styrene-acrylamide-acrylic acid) microspheres [see Fig. 5(a)], QDs [see Fig. 5(b)], and QDsAAM [see Fig. 5(c)]. In Fig. 5(a), the wide peak at 3462 cm−1 is assigned to the superimposed stretching vibrations of O–H and N–H bonds, while the strong peak at 1652 cm−1 is attributed to the C=O bond vibration. These data confirm the presence of carboxyl groups and amino groups on the microspheres. In Fig. 5(b), the strong peak at 3468 cm−1 represents the superimposed stretching vibrations of the O–H and N–H bonds, while the strong peak at 1650 cm−1 represents the characteristic absorption peak of the C=O bond. The illustration in Fig. 5(b) confirms the presence of aminoacetic acid, which has been used to modify the surface of QDs. In Fig. 5(c), the wide peak at 3434 cm−1 is similar to the 3462 cm−1 and 3468 cm−1 in peaks in Fig. 5(a) and (b). Similarly, the stronger wide adsorption peak at 1666 cm−1 is similar to both the 1652 cm−1 and 1658 cm−1 peaks in Fig. 5(a) and (b).
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Fig. 7. Linear correlation between QDsAAM’s concentration and fluorescence intensity.
Fig. 6. Fluorescence emission spectra of (a) QDs (dissolved in ethanol), (b) QDsAAM aqueous suspension, (c) hydrazide QDsAAM aqueous suspension, (d) protein-immobilized QDsAAM aqueous suspension, and (e) poly(styrene-acrylamide-acrylic acid) microspheres. All the aqueous suspensions were set at concentration of 35.0 × 10−3 g·L−1 and pH value of 7.0. All measurements were taken at 20 ◦ C. IF refers to the fluorescence intensity. TABLE I EXCITATION AND EMISSION WAVELENGTH OF THE SAMPLES
D. Fluorescent Characteristics The respective fluorescence spectra of QDsAAM, hydrazide QDsAAM, protein- immobilized QDsAAM, and poly(styreneacrylamide-acrylic acid) microspheres without QDs are shown in Fig. 6. QDsAAM, hydrazide QDsAAM, and proteinimmobilized QDsAAM can give off fluorescence when properly excited. The maximum excitation and emission wavelength of the samples are listed in Table I. The poly(styrene-acrylamideacrylic acid) microspheres have a slight emission at the wavelength of 306 nm. The fluorescence that comes from the poly(styrene-acrylamide-acrylic acid) was much different from QDsAAM’s at emission wavelength and much weaker in fluorescence intensity. This indicates that the fluorescence of QDsAAM comes from the inside QDs. Compared with pure QDs, an obvious blue shift was observed in the QDsAAM together with the broadening of full-width at half maximum (FWHM). We thought that the high concentration
Fig. 8. Effect of pH on fluorescence intensity. The aqueous suspension was set at a concentration of 25 × 10 −3 g·L−1 .
of QDs embedded in the inner layer will make FWHM broaden to some extent [21]. At the same time, we speculate that the interaction between the QDs and microspheres, and the difference in refractive index caused by the two layers and the thick outer layer are the main reasons for the dramatic blue shift [22], [23]. It is noted that no change in emission wavelength was found after hydrazide reaction, and protein immobilizations were performed. The probable explanation could be that hydrazide reaction and protein immobilization were processed mostly on the surface of the microspheres, which did not cause changes in the QDs inside. The quantum yield of QDsAAM was measured as 21%, which was depressed as compared to pure QDs reported elsewhere [19]. At the same time, the UV/Vis’ absorption of poly(styrene-acrylamide-acrylic acid) without QDs was detected, and slight absorption was found in the UV area, which was little compared with the QDsAAM absorption. The pH value and salt concentration are important environment factors in biological detection. The effect of pH on fluorescence was investigated through gradual variation of the pH value from 1 to 14. As can be seen from Fig. 8, fluorescence intensity changes according to the pH value. The microspheres were well dispersed during the pH variation, and the fluorescence character change mainly focused on fluorescence intensity, with no shift being found on the emission wavelength. This could possibly indicate that there was no physical or structural alteration
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shown). The results indicated that the embedment of QDs into the polymer matrix could protect the QDs from environmental interactions and could also exert some influence on the fluorescence characteristics. Moreover, there was no significant change in the fluorescent characteristics after QDsAAM was stored for six months. E. Immobilization of BSA Onto Azidocarbonyl QDsAAM
Fig. 9. Effect of electrolyte concentration on fluorescence intensity (pH 7.0). The aqueous suspension was set with a concentration of 15 × 10−3 g·L−1 .
in the inner fluorescent material QDs. It is worth noting that fluorescence intensity has strengthened with the system’s pH value increasing within the pH range from 1 to 12. This could be because the excessive H+ ions in the aqueous solution have a slight effect on the 3-D structure of the microspheres. Moreover, we can see from the results that the polymer matrix can protect the QDs with lower pH value from aggregation. This makes it possible for QDs to be used in acidic conditions. The effect of ionic strength adjusted with NaCl (ranging from 0.1 to 0.5 mol·L−1 ) of QDsAAM is illustrated in Fig. 9. As can be seen, the same phenomenon occurred with pH variation in that no aggregation was found, and emission wavelength demonstrated the result of no shift. In addition, it could be noted that fluorescence intensity has a slight decrease, which could be due to the Coulombic interaction between microspheres becoming weak as the ionic strength increases. The scattering effect in high ionic concentration would also be an explanation. The linear correlation between the microspheres’ concentration and fluorescence intensity could provide opportunity for semiquantitative detection when the targets such as antigen or antibody are linked onto the QDsAAM surface. The linearity between QDsAAM and its fluorescence intensity was investigated and found to remain in a broad range from 3.0 × 10−3 to 90.0 × 10−3 g·L−1 (see Fig. 7). The embedment of QDs into the polymer matrix makes it impossible for QDs to agglomerate in bulk. The stable distribution of QDs into the polymer provides an opportunity for the concentration and fluorescence intensity to increase linearly. The linear regression equation is Y = 15287540X − 54791.01 (R2 = 0.9944), which shows that these microspheres could be used in subtle detections. Here, Y represents fluorescence intensity and X stands for the concentration with units of grams per liter. The reason why linearity was limited in a certain range could be due to the inner-filter effect from the concentration of the microspheres themselves. At the same time, we also investigated the effect of temperature on the characteristics of fluorescence. In biology, according to the application’s need, we detected the fluorescence characteristic change to be between 20 ◦ C and 40 ◦ C. And no obvious change was found when the temperature increased (data not
Protein immobilization on various supports has been widely used in many areas such as solid-phase diagnostics, biosensors, and bioseparation. Such immobilization requires the support of functional groups suitable for coupling. In previous studies, the diazotized polystyrene latex could be covalently coupled with phenol and imidazole groups of antibodies without any crosslinking agent [24]. This method for protein immobilization has advantages in terms of speed, sensitivity, very high specificity, and relatively high binding capacity (20%–40%). The amide groups were converted into the azidocarbonyl groups through a two-step simple reaction, while BSA was covalently combined with the azidocarbonyl microspheres by peptide bonds at a low temperature. As measured, the immobilized capacity was 40 × 10−3 g/g, indicating that the azidocarbonyl microspheres possessed high protein immobilization capacity. After protein immobilization, the linearity relation was kept at Y = 13174280X − 24751.24 (R2 = 0.9931). It should be noted that fluorescence intensity has decreased slightly as compared to QDsAAM after protein immobilization. This is probably because the BSA on the surface absorbs the light coming through the polymer matrix. IV. CONCLUSION In this paper, double-layer structure QDs-tagged microspheres were synthesized, suggesting high trapping ratio and little leakage. Azidocarbonyl groups were converted from amido groups on the surface, and this allowed convenient immobilization of protein. The linear relationship between fluorescence intensity and concentration revealed their potential quantitative measurements for application in biomedical research. ACKNOWLEDGMENT The authors would like to thank Prof. D. Pang for providing the QDs. REFERENCES [1] J. Qian, J. Truebenbach, F. Graepler, P. Pereira, P. Huppert, T. Eul, G. Wiemann, and C. Claussen, “Application of poly-lactide-co-glycolidemicrospheres in the transarterial chemoembolization in an animal model of hepatocellular carcinoma,” World J. Gastroenterol., vol. 9, no. 1, pp. 94–98, Jan. 2003. [2] X. D. Cao and M. S. Shoichet, “Delivering neuroactive molecules from biodegradable microspheres for application in central nervous system disorders,” Biomaterials, vol. 20, no. 4, pp. 329–339, Feb. 1999. [3] J. P. Benoit, N. Faisant, M. C. V. Julienne, and P. Menei, “Development of microspheres for neurological disorders: From basics to clinical applications,” J. Control. Release, vol. 65, no. 1–2, pp. 285–296, Mar. 2000. [4] S. Phadtare, A. Kumar, V. P. Vinod, C. Dash, D. V. Palaskar, M. Rao, P. G. Shukla, S. Sivaram, and M. Sastry, “Direct assembly of gold nanoparticle “Shells” on polyurethane microsphere “Cores” and their application
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[5]
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Xinghua Pan received the B.E. degree in biomedical engineering in 2007 from Xi’an Jiaotong University, Xi’an, China, where he is currently working toward the M.E. degree at the Key Laboratory of Biomedical Information Engineering of the Ministry of Education. He is currently engaged in the synthesis and application of functional fluorescent and magnetic particles.
Maolin Lu was born in Anhui, China, in 1982. She received the B.S. degree in bioengineering in 2006 from Xi’an Jiaotong University, Xi’an, China, where she is currently working toward the M.S. degree. Her current research interests include synthesis, analysis, and application of fluorescent nanoparticles.
Daocheng Wu was born in Zhenjiang, China, in 1962. He received the M.S. degree in applied chemistry from the Changsha Institute of Technology, Changsha, China, in 1989, and the Ph.D. degree in biomedical engineering from Xi’an Jiaotong University, Xi’an, China, in 2003. During 2004, he was a Professor in the School of Life Science and Technology, Xi’an Jiaotong University, where he is currently an Associate Dean of the School of Life Science and Technology. His current research interests include biomaterials, bionanotechnology, and related topics that include the drug delivery system, nanoparticles for diagnosis and therapy, as well as functional nanoparticles and their applications in the biomedical field.
Lili Gai was born in Heilongjiang, China, in 1984. She received the B.S. degree in bioengineering in 2006 from Xi’an Jiaotong University, Xi’an, China, where she is currently working toward the M.S. degree. Her current research interests include the synthesis of intelligent microspheres carriers and their application in biotechnology.
