Biomaterials 32 (2011) 1177e1184
Contents lists available at ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
A multicomponent recognition and separation system established via fluorescent, magnetic, dualencoded multifunctional bioprobes Jun Hu a, Min Xie a, Cong-Ying Wen a, Zhi-Ling Zhang a, Hai-Yan Xie b, An-An Liu a, Yong-Yong Chen c, Shi-Ming Zhou c, Dai-Wen Pang a, * a
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, and State Key Laboratory of Virology, Wuhan University, Wuhan 430072, PR China School of Life Science and Technology, Beijing Institute of Technology, Beijing 100081, PR China c Surface Physics Laboratory (National Key Laboratory), Department of Physics, Fudan University, Shanghai 200433, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 August 2010 Accepted 6 October 2010 Available online 10 November 2010
Accurate and rapid recognition and separation of multiple types of biological targets such as molecules, cells, bacteria or viruses from complex sample mixtures is of great importance for a wide range of diagnostic and therapeutic strategies. To achieve this goal, a set of fluorescent, magnetic, dual-encoded multifunctional bioprobes has been constructed by co-embedding different-sized quantum dots and varying amounts of g-Fe2O3 magnetic nanoparticles into swollen poly(styrene/acrylamide) copolymer nanospheres. The dual-encoded bioprobes, which possessed different photoluminescent property and magnetic susceptibility, were proven to be capable of simultaneously recognizing and separating multiple components from a complex sample when three kinds of lectins were used as the targets. The lectins were separated with high efficiency and kept their bioactivity during the process. Compared to the conventional batchwise separation, this method does not require a large number of sequential reaction steps, which is economical of time and can be very reagent-saving. By combining the multiplexing capability of quantum dots with the superparamagnetic properties of iron oxide nanoparticles, this dual-encoded technique is expected to open new opportunities in high-throughput and multiplex bioassays, such as cell sorting, proteomical and genomical applications, drug screening etc. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Fluorescence Magnetism Nanocomposite Biosensor
1. Introduction Multiplex and high-throughout assays are increasing in their popularity with researchers as an essential tool for diagnosis, drug screening, gene expression etc. Compared with conventional assay technologies, such as planar arrays [1e3], the bead-based suspension assays show fast binding kinetics and high flexibility [4e12]. Trau et al. described two approaches to fabricate optically encoded microspheres embedded with fluorescent dyes for high-throughput screening applications in drug discovery and genomics [4]. However, polymeric beads optically encoded with organic dyes have many limitations, such as photobleaching and narrow excitation spectra of dyes. Quantum dots (QDs), a fluorescent semiconductor nanocrystal widely used in biological recognition and labeling, have
* Corresponding author. Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, and State Key Laboratory of Virology, Wuhan University, Wuhan 430072, PR China. Tel.: þ86 2768756759; fax: þ86 27 68754067. E-mail address:
[email protected] (D.-W. Pang). 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.10.015
unique optical property, such as color-tunable emission in wide spectral range with simultaneous excitation, which makes them show great potential in optical encoding. By embedding QDs with different emission wavelength into polymer spheres, a large number of encoded and distinguishable microbeads can be obtained, which are expected to be used in gene expression, high-throughput screening, and medical diagnostics [5e7]. Wilson et al. described a simple method for preparing encoded microspheres by assembling hydrophobic CdSe/ZnS quantum dots on polyamine-coated microspheres in chloroform [8]. Li and Sukhanova et al. showed the possibility to obtain hundreds of codes by depositing differentwavelength QDs on surfaces of silica colloidal crystals beads, and demonstrated the application of the nanocrystal-encoded microbeads for DNA assay and proteomics [9,10]. The capability to separate specific biological targets from complex samples with high efficiency and high throughput is of great importance for a wide range of biological applications, such as in vitro diagnostic and cell-based therapy. But the conventional batchwise separation processes require a large number of sequential reaction steps, which are very time-consuming. To overcome this
1178
J. Hu et al. / Biomaterials 32 (2011) 1177e1184
limitation, a number of separation methods based on magnetic encoding have been investigated. Magnetic particles with different size could be separated from each other according to the different response under certain magnetism [13], which could be used for magnetic encoding. Adams et al. made use of microfluidic technology to achieve simultaneous spatially-addressable sorting of multiple cell types labeled with different magnetic tags in a continuous-flow manner [14]. Because of the complexity of biological samples, the demand on the strategy used to make simultaneous detection and separation of multiple components became more critical. Take the example of cell analysis, a number of cells should be simultaneously identified in one-pot reaction and sometimes they should also be selectively isolated from the mixture at the same time for further research, such as surface protein or DNA assay of each cell line. However, encoded spheres with only fluorescence or magnetism could not meet this demand. We have previously developed a new strategy to fabricate multifunctional nanospheres with both fluorescence and magnetism with high structural stability by co-embedding quantum dots and nano-g-Fe2O3 into poly(styrene/acrylamide) copolymer nanospheres [15e19]. Here, based on this technology, a set of fluorescent, magnetic, dual-encoded spheres was constructed and functionalized to simultaneously recognize and selectively separate three kinds of lectins (Dolichos biflorus agglutinin (DBA), peanut agglutinin (PNA) and Ulex europaeus agglutinin I (UEA I)) from complex samples with high efficiency. The principle of this method could easily be extended to other uses, such as cells category, proteomics, genomics etc, depending on the tags on the surface of encoded spheres. 2. Materials and methods 2.1. Reagents and instruments Anti-Dolichos biflorus agglutinin (Anti-DBA), anti-peanut agglutinin (Anti-PNA), anti-Ulex europaeus agglutinin I (Anti-UEA I), fluorescein-labeled Dolichos biflorus agglutinin (F-DBA), peanut agglutinin (F-PNA) and Ulex europaeus agglutinin I (FUEA I) were purchased from Vector laboratories (Burlingame, USA). The carboxyl terminal magnetic spheres with 3-mm diameter (proMag 3 series COOH surfactantfree microspheres) were obtained from Bangs Laboratories. Other chemical reagents were of analytical grade and used as supplied without further purification. Fluorescence images were recorded with a Nikon ECLIPSE TE2000-U inverted fluorescence microscope equipped with a Nikon INTENSILIGHT C-HGFI lamp and Q-IMAGING RETIGA 2000R CCD. A Fluorolog-3 (HORIBA JOBIN YVON) fluorescence spectrometer was used to record the fluorescence signals of fluorescein and fluorescent nanospheres. UVeVis absorption spectrum was performed on a UV-2550 UVeVis spectrophotometer from Shimadzu Corporation. The magnetic hysteresis loops were measured with a Lakeshore 7407 vibrating sample magnetometer (VSM).
magnetic nanospheres and yellow-fluorescent nanospheres were suspended in 0.01 M phosphate buffer solution (PBS) in a test tube, which were then selectively separated with the assistance of an extra-applied magnetic field as illustrated in Scheme 1. Briefly, after the mixed solution was put onto a magnetic scaffold for about 10 s, the 3-mm-diameter spheres with strong-magnetism were attracted, while the suspensions were removed to another tube which was subsequently allowed for another 3-min attraction. Then, the nonmagnetic YFS was removed, with the RFMS remained. At last, all of the three kinds of spheres separated were respectively washed five times by magnetic attraction and fluorescence spectra were recorded by fluorescence spectrometer. 2.4. Antibody immobilization Approximately, 0.4 mg of Anti-PNA and Anti-UEA I were oxidized with periodic acid according to a previously published method to create active aldehydes [16]. The aldehyde-containing Anti-PNA and Anti-UEA I were then respectively reacted with 200 mL of amino-terminated RFMS (w3 mg) and YFS (w3 mg) in 0.5 mL of phosphate buffer solution (PBS) for more then 6 h at 25 C with gentle shaking. After washing 5 times with 0.01 M PBS (pH 6.8) by centrifugation, Anti-PNA-modified red-fluorescent-magnetic nanospheres (Anti-PNA-RFMS) and Anti-UEA I-modified yellowfluorescent nanospheres (Anti-UEA I-YFS) were constructed. At the same time, 10 mL (w0.26 mg) of 3-mm-diameter magnetic spheres (MS) with carboxyl terminal, after washing twice with 0.1 M PBS (pH 6.8), were activated with 5 mg of 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDAC) in 0.4 mL of 0.1 M PBS (pH 6.8) for 30 min at room temperature with continuous shaking. Then, 0.2 mg of Anti-DBA was incubated with the activated magnetic microspheres for 4 h at room temperature to form Anti-DBA-modified magnetic microspheres (Anti-DBA-MS). After removing unreacted antibodies, these resulting spheres were stored in 200 mL 0.01 M PBS (pH 7.2) at 4 C. 2.5. Capture capability of fluorescent, magnetic, dual-encoded spheres To evaluate the capture capability of the prepared fluorescent, magnetic, dualencoded spheres, fluorescein-labeled lectins were used. 40 mL of antibody-modified spheres (Anti-DBA-MS, Anti-PNA-RFMS and Anti-UEA I-YFS) were incubated with excess F-DBA, F-PNA and F-UEA I respectively in 500 mL of 0.01 M PBS (pH 7.2) for 30 min at room temperature with gentle shaking. After separation with a magnetic scaffold (for MS and RFMS) or by centrifugation (for YFS) and washing 5 times with PBS, the capture capacity of the prepared spheres to corresponding lectins was calculated by using the fluorescein assay mentioned previously [19]. 2.6. Specificity of fluorescent, magnetic, dual-encoded spheres In order to confirm the bioactivity and specificity of antibodies on the surfaces of these as-prepared multifunctional spheres, 100 mL of Anti-PNA- and Anti-UEA I-
2.2. Preparation of fluorescent, magnetic, dual-encoded spheres By a modified method of emulsifier-free polymerization, poly(styrene/acrylamide) copolymer nanospheres were prepared with hydrophobic hollow cavities [15]. Then, CdSe/ZnS quantum dots with fluorescence emission peak at 610 nm and a mount of nano-g-Fe2O3 magnetic particles were co-embedded into single swelling nanospheres to fabricate red-fluorescent-magnetic nanospheres (RFMS). With the same method, yellow-fluorescent nanospheres (YFS) were prepared using CdSe/ZnS quantum dots with 574 nm fluorescence emission peaks. Combining with the 3-mm diameter magnetic spheres (MS), a set of optically and magnetically encoded spheres with different emission wavelength and magnetic characteristics was constructed. 2.3. Feasibility for fluorescent-magnetic codes To investigate the differences of magnetic response of the MS and RFMS, a series of centrifuge tubes respectively containing two kinds of magnetic spheres were put onto a magnetic scaffold for a certain time (0, 5, 10, 15, 20, 25, 30 s for MS and 0, 10, 30, 60, 120, 180 s for RFMS). Then, the suspensions in each tube were drawn out to measure the absorbance at 600 nm, from which the capture efficiency of the magnetic spheres at different times was obtained. The performance of the optically and magnetically encoded spheres was initially characterized with a mixture of MS, RFMS and YFS in order to optimize the separation conditions. A ternary mixture of 3-mm-diameter magnetic spheres, red-fluorescent-
Scheme 1. Selective separation of three kinds of spheres (magnetic spheres with 3 mm diameter (MS), red-fluorescent-magnetic nanospheres (RFMS) and yellow-fluorescent nanospheres (YFS)) with the assistance of an external magnetic field.
J. Hu et al. / Biomaterials 32 (2011) 1177e1184 modified red-fluorescent-magnetic nanospheres fabricated by the method mentioned above were respectively incubated with 100 mL of fluorescein-labeled PNA and UEA I (0.05 mg/mL) in 500 mL 0.01 M PBS (pH 7.2) for 30 min. Similarly, 100 mL of Anti-DBA-modified 3-mm-diameter magnetic spheres were reacted with 10 mL of fluorescein-labeled DBA (0.05 mg/mL) for 30 min with gentle shaking. As a control, 100 mL of Anti-DBA-modified 3-mm-diameter magnetic spheres were mixed with 100 mL of F-PNA (0.05 mg/mL) or 100 mL of F-UEA I (0.05 mg/mL) to check whether there were some cross-reaction between them, the same method was also performed on Anti-PNA-RFMS and Anti-UEA I-modified RFMS (Anti-UEA I-RFMS). After a fifth wash, these resulting spheres were subsequently observed with a microscope. 2.7. Recognition and separation of lectins 200 mL of each antibody-modified spheres (Anti-DBA-MS, Anti-PNA-RFMS and Anti-UEA I-YFS) were added into a mixed solution consisting of 10 mL of F-DBA (0.05 mg/mL), 100 mL of F-PNA (0.05 mg/mL) and 100 mL of F-UEA I (0.05 mg/mL), which were then concentrated to 500 mL in 0.01 M PBS (pH 7.2). After 30 min incubation, the final products were selectively separated with the assistance of an extra-applied magnetic field as described in Scheme 1. After the separation, the fluorescence signals of fluorescein-lectins-combined spheres were obtained and the separation efficiency of target lectins were calculated by the fluorescein assay mentioned previously [19]. 2.8. Biorecognition of glycoconjugates To make sure that the lectins separated were bioactive, the glycoconjugates biorecognition experiments were carried out as described below. A549 cells were seeded onto a 24-well tissue culture plate and grown in DMEM medium with 10% fetal bovine serum, 100 U/mL penicillin G sodium, 0.1 g/L streptomycin sulfate at 37 C under 5% CO2. After the cells became confluent, they were washed twice with 1 mL of 1 PBS, and 200 mL of lectin-combined RFMS and YFS isolated above were added into separate cell wells, respectively. Then A549 cells were incubated for 30 min with gentle shaking at room temperature. Redundant particles were removed by washing twice with 1 mL of 1 PBS, and the A549 cells sticking in the wells were added with culture media and subsequently observed with a microscope. Taking account of the strong nonspecific adsorption of 3-mm-diameter magnetic spheres to the tissue culture plate, 200 mL of DBA-combined MS separated above were incubated with suspension A549 cells detached by trypsin-EDTA solution for 30 min. After that, the cells were captured and washed twice with a magnetic scaffold. Fluorescence images were obtained by fluorescence microscope.
3. Results and discussion 3.1. Feasibility for fluorescent-magnetic dualcodes
1179
colors or emission peaks under excitation, and used to encode thousands or even millions of genes, proteins, and small-molecule compounds. Magnetic particles, a kind of material for separation, become magnetized and are hence dragged at an external magnetic field. In a certain solution, the magnetically induced flow velocity on the particle is proportional to their magnetic susceptibility. Thus, magnetic particles that are different in their magnetic susceptibility could be captured in turn by an externally applied field and separated from each other. Combining the multiplexing capability of quantum dots and magnetic particles, a set of fluorescent, magnetic, dual-encoded spheres (magnetic spheres with 3 mm diameter (MS), red-fluorescent-magnetic nanospheres (RFMS) with 200 nm diameter and yellow-fluorescent nanospheres (YFS) with 200 nm diameter) was constructed. The magnetic hysteresis loops of 3-mm magnetic spheres and red-fluorescent-magnetic nanospheres were measured at room temperature as shown in Fig. 1, from which it can be seen that both of these two kinds of magnetic spheres exhibited superparamagnetic characteristics at room temperature. The magnetic saturation value of MS was calculated to be 27.6 emu/g, which was about three times larger than that of RFMS. It implied that these two kinds of magnetic spheres could be separated from each other under certain conditions. The feasibility for magnetic codes of the as-prepared spheres was proved by their different response under an external magnetic field, which was generated by a magnetic scaffold. Magnetic scaffold is a magnetic particle concentrator and in the central part there are two pieces of NdeFeeB magnets, which was coated with a plastic shell (about 2-mm thick). A two-dimension numerical simulation of the magnetic field gradients for the magnets was shown in Fig. 2 and the surface field strength of the magnetic scaffold was about 0.4 T, which was large enough to magnetize these two kinds of magnetic particles. After the centrifuge tubes respectively containing MS or RFMS were put onto a magnetic scaffold for a certain time, the suspensions in each tube were drawn out to measure the absorbance at 600 nm. As the absorbance at 600 nm was proportional to the concentration of spheres (as shown in Fig. 5), the capture efficiency of MS and RFMS at different times could be calculated by Eq. (1):
Encoded technology is widely used as a method for simultaneous analysis of multiple components. The central part is constructing a series of materials with different tags, such as fluorescent or magnetic. Varied-sized quantum dots could be regarded as a set of fluorescent codes because of their different
4T ¼ ð1 AT =A0 Þ 100%
Fig. 1. Magnetic hysteresis loops of 3-mm magnetic spheres (square) and red-fluorescent-magnetic nanospheres (triangle).
Fig. 2. Numerical simulation of the magnetic field gradients for the magnets in the magnetic scaffold. The size for each magnet was 19-mm long, 6-mm high and 12-mmwide.
(1)
where 4T is the capture efficiency of spheres at different times, AT is the absorbance of suspensions at 600 nm after the spheres were
1180
J. Hu et al. / Biomaterials 32 (2011) 1177e1184
captured for a certain time, A0 is the absorbance of original spheres solutions at 600 nm. As shown in Fig. 3A, nearly all the 3-mm-diameter magnetic spheres could be captured by 10-s attraction. At the same condition, there were only 12.05% of red-fluorescent-magnetic nanospheres captured (Fig. 3B). (However, during the practical mixture separation, only 3-mm-diameter magnetic spheres left while they were further washed by another 10-s attraction for several times.) After 2 min, all of the RFMS could be captured by the magnetic scaffold (Fig. 3B), while the nonmagnetic yellow-fluorescent nanospheres had no response at this condition. So, according to their different actions under the external magnetic field, these three kinds of spheres could be used as magnetic codes for selective separation of multiple types of biological targets from complex mixtures. On the other hand, the different fluorescent emissions of embedded quantum dots enabled them with the power to simultaneously recognize different targets in complex sample. Therefore, a set of spheres with both fluorescent and magnetic codes was constructed. As expected, according to the separation method described in Scheme 1, 3-mm-diameter magnetic spheres, redfluorescent-magnetic nanospheres and yellow-fluorescent nanospheres could be separated in turn from the ternary mixture, which was shown in Fig. 3C and D. Compared with the double hump of the ternary mixture in fluorescent spectrum, the as-separated spheres showed different fluorescent signal as shown in Fig. 3C: no emission for MS, a fluorescent emission peak of red-QDs at 610 nm for RFMS and yellow-QDs at 574 nm for YFS, respectively. There was only one kind of spheres could be seen at each batch under
a microscope with a 100 oil-immersion objective lens (Fig. 3D), which further confirmed that these as-separated multifunctional spheres were pure without mixing with each other and indicated that these three kinds of spheres could be the ideal fluorescently and magnetically encoded materials for rapid separation. 3.2. Specificity of antibody-modified fluorescent, magnetic, dualencoded spheres We subsequently examined the performance of dual-encoded spheres for recognition and separation of multiple components by using fluorescein-labeled Dolichos biflorus agglutinin (F-DBA), peanut agglutinin (F-PNA) and Ulex europaeus agglutinin I (F-UEA I). Before that, these dual-encoded spheres were respectively modified with anti-Dolichos biflorus agglutinin (Anti-DBA), antipeanut agglutinin (Anti-PNA), anti-Ulex europaeus agglutinin I (Anti-UEA I). To characterize the bioactivity and specificity of antibodies on the surfaces of the spheres, Anti-DBA-modified MS (Anti-DBA-MS), Anti-PNA-modified RFMS (Anti-DBA-RFMS) and Anti-UEA I-modified RFMS (Anti-UEA I-RFMS) were incubated with F-DBA, F-PNA and F-UEA I separately. After removing the unbound lectin molecules by centrifugation or magnetic attraction, the spheres were observed with a fluorescence microscope. Microscopic analysis of the green fluorescence clearly demonstrated the binding of lectins to the corresponding antibodies on the surfaces of the spheres (Fig. 4A,E and I), indicating that the activity of the antibodies was preserved during the coupling process. On the other hand, there
Fig. 3. Capture efficiency of 3-mm-diameter magnetic spheres (A), red-fluorescent-magnetic nanospheres (B) at different times, and the fluorescence spectra of the ternary mixture of 3-mm-diameter magnetic spheres, red-fluorescent-magnetic nanospheres and yellow-fluorescent nanospheres before and after separation (C), accompanied by the microscopic images (D).
J. Hu et al. / Biomaterials 32 (2011) 1177e1184
1181
was nearly not any visible fluorescence of fluorescein when AntiDBA-MS were incubated with F-PNA (Fig. 4B) or F-UEA I (Fig. 4C). Similarly, no green fluorescence signal was detected when AntiPNA-RFMS were incubated with F-DBA (Fig. 4D) or F-UEA I (Fig. 4F) and it was the same case when Anti-UEA I-modified spheres were mixed with F-DBA (Fig. 4G) or F-PNA (Fig. 4H). These results demonstrated that the multifunctional spheres could specifically recognize corresponding lectins through their surface antibodies under the conditions. 3.3. Capture capacity of antibody-modified spheres Capture capacity of as-prepared fluorescent, magnetic, dualencoded spheres to corresponding lectins was an important parameter in applications. So, fluorescence-based quantitative analysis was used to evaluate the amount of lectins combining with the antibody-modified spheres (Anti-DBA-MS, Anti-PNA-RFMS and Anti-UEA I-modified YFS (Anti-UEA I-YFS)). After correspondingly incubated with excess F-DBA, F-PNA and F-UEA I, the antibodymodified spheres were separated with magnetic scaffold (for MS and RFMS) or by centrifugation (for YFS) and washed 5 times with phosphate buffer solution (PBS). The binding capacity of spheres to corresponding lectins was calculated as follows. As mentioned above, the absorbance of these three kinds of spheres at 600 nm was proportional to the concentration. So, it was possible to obtain the concentrations of the spheres after the
Fig. 5. Concentration dependence of absorbance intensity for MS, RFMS and YFS at 600 nm.
binding of lectins based on the calibration curve (Fig. 5), which was constructed by measuring the absorbance of unmodified-spheres with known amounts at 600 nm. In order to evaluate the amounts of lectins binding on the surface of spheres, fluorescence-based
Fig. 4. Fluorescent microscopic images of three kinds of antibody-modified spheres incubated with different fluorescein-labeled lectins. (A-C, Anti-DBA-MS incubated with F-DBA, F-PNA and F-UEA I respectively, D-F, Anti-PNA-RFMS incubated with F-DBA, F-PNA and F-UEA I separately, G-I, Anti-UEA I-RFMS incubated with F-DBA, F-PNA and F-UEA I respectively.)
1182
J. Hu et al. / Biomaterials 32 (2011) 1177e1184
antibody-modified spheres was calculated to be of 2.4 mg F-PNA/ 1 mg RFMS and 2.8 mg F-UEA I/1 mg YFS respectively. 3.4. Performance of fluorescent, magnetic, dual-encoded spheres for simultaneous recognition and separation of lectins
Fig. 6. Fluorescent calibration curve of three different fluorescein-labeled lectins: fluorescence signal of fluorescenin-labeled lectins at different concentrations were recorded in the presence of unmodified-spheres at certain amounts.
quantitative method was used [19]. The fluorescence signal of fluorescenin-labeled lectins at different concentrations was recorded with a fluorescence spectrometer respectively to get the standard curves of fluorescein (Fig. 6). Taking account of the effect of iron oxide on fluorescence signal [20], the calibration curves were constructed at the presence of unmodified-spheres at a fixed concentration equal to that of isolated lectin-bound spheres. Then, the amount of fluorescein-labeled lectins immobilized on the spheres can be obtained based on the standard curve of fluorescein and the fluorescence spectra of fluorescein-labeled lectin-bound spheres. So, the capture capacity of antibody-modified MS for DBA was determined to be about 2.2 mg F-DBA/1 mg MS, while the amounts of F-PNA and F-UEA I on the surface of corresponding
For simultaneous recognition and separation of these three kinds of lectins, fluorescent, magnetic, dual-encoded multifunctional spheres (Anti-DBA-modified strong-magnetism microspheres with no fluorescence, Anti-PNA-modified red-fluorescentmagnetic nanospheres with weak magnetism and Anti-UEA Imodified yellow-fluorescent nanospheres without nano-g-Fe2O3 embedded) were incubated with the mixed solution containing FDBA, F-PNA and F-UEA I. After incubation, these dual-encoded spheres were separated from each other by the means of an external magnetic field for 10-s and then 3-min attraction as described in Scheme 1. Fluorescence images were then recorded with fluorescence microscope with blue-light excitation. The green fluorescence of fluorescein on the surface of spheres (Fig. 7A,C and E) showed the interaction between lectins and fluorescent, magnetic, dual-encoded spheres. To confirm the effectivity of this separation method, the corresponding fluorescence spectra of these as-separated multifunctional spheres were recorded by fluorescence spectrometer. After sorting, the fluorescence spectra showed the emission of fluorescein with no QDs (Fig. 7B), red-QDs at 610 nm (Fig. 7D) and yellow-QDs at 574 nm (Fig. 7F), respectively. From which, we could conclude that the fluorescent, magnetic, dual-encoded spheres were successfully used for simultaneous detection and selective separation of these three kinds of lectins with high efficiency. 3.5. Separation efficiency of target lectins Combining with the fluorescent calibration curve of fluoresceinlabeled lectins constructed by the same method mentioned above and the absorbance of multifunctional spheres at 600 nm, the
Fig. 7. Fluorescent microscopic images of the separated fluorescent, magnetic, dual-encoded spheres: (A) Anti-DBA-MS combined with F-DBA, (C) Anti-PNA-RFMS combined with F-PNA and (E) Anti-UEA I-YFS combined with F-UEA I. B, D and F were the corresponding fluorescence spectra.
J. Hu et al. / Biomaterials 32 (2011) 1177e1184
1183
Fig. 8. Microscopic images for recognition A549 cells by (A, B) Anti-DBA-MS combined with F-DBA, (C, D) Anti-PNA-RFMS combined with F-PNA and (E, F) Anti-UEA I-YFS combined with F-UEA I, (top right corner in B: microscopic images of single cell with a 100 oil-immersion objective lens). A, C, E were fluorescent images, while B, D, F were bright field images.
separation efficiency of encoded spheres and corresponding lectins were obtained as illustrated in Eqs. (2) and (3):
4S ¼ ðAS =AI Þ 100%
(2)
4L ¼ ðCS =CI Þ 100%
(3)
where 4S and 4L are the separation efficiency of spheres and lectins, AS and AI are the absorbance of spheres separated and initially added at 600 nm with the same volume, CS and CI are the concentrations of lectins separated and initially added at the same volume, which were obtained from the fluorescence spectra and calibration curve of fluorescein-labeled lectins. It should be noted that the separation efficiency of Anti-DBA-MS to DBA was the highest which was calculated to be about 53.0%, compared with the separation efficiency of Anti-PNA-RFMS to PNA which was 39.0% and that of AntiUEA I-YFS to UEA I which was 33.2%. The separation efficiency of proteins will depend on the recovery ratio of corresponding spheres. Because of the loss of multifunctional spheres during the separating and washing steps, the separation efficiency of target lectins was not
so high. As we calculated from the absorbance of multifunctional spheres at 600 nm, the recovery ratios of MS, RFMS and YFS were only 62.0%, 50.0% and 35.2% respectively.
3.6. Bioactivity of lectins after separation DBA with two carbohydrate-binding sites are specific for Nacetylgalactosamine residues. The two binding sites of PNA are specific for D-galactosamine and UEA I can recognize L-fucose specifically. A549 cells with these three kinds of glycoconjugates expressed on surface were incubated with lectin-combined spheres separated above to confirm the bioactivity of lectins. As shown in Fig. 8A, it was clearly to see the green fluorescence spheres on the surfaces of A549 cells when the separated MS were incubated with suspension cells for about 30 min. Compared with that of MS unmodified, no cell could be captured (data not shown). It was concluded that the fluorescence derived from the interaction between lectins and A549 cells and that the F-DBA separated were bioactive. Similarity in the RFMS combined with F-PNA and YFS
1184
J. Hu et al. / Biomaterials 32 (2011) 1177e1184
combined with F-UEA I, fluorescence of spheres was obvious on the surfaces of A549 cells sticking in the wells (Fig. 8C and E), which demonstrated that the bioactivity of lectins was preserved during the separation process. 4. Conclusions A set of fluorescent, magnetic, dual-encoded multifunctional bioprobes was constructed with a simple and convenient method, which could be used in real-time detection and simultaneous separation of multiple targets. After modified with anti-Dolichos biflorus agglutinin, anti-peanut agglutinin and anti-Ulex europaeus agglutinin I respectively, these biofunctional spheres were successfully used to recognize and separate DBA, PNA and UEA I simultaneously, which was very time-saving and could reduce the consumption of reagent and avoid tedious manual handling. The lectins separated were bioactive, which could specifically recognize the surface-expressed glycoconjugates of A549 cells. This strategy could also be applied in many areas of biochemical analysis, such as stem cell recognition and separation or high-throughput screening in drug discovery, both of which could lead to new possibilities in the treatment of various diseases. Acknowledgements This work was supported by the National Key Scientific Program (973)-Nanoscience and Nanotechnology (Nos. 2006CB933100 and 2011CB933600), the Science Fund for Creative Research Groups of NSFC (Nos. 20621502 and 20921062), the National Natural Science Foundation of China (Grant Nos. 20875072, 20833006 and 81071227), the Ministry of Public Health (Nos. 2009ZX10004-107 and 2008ZX10004-004) and Fundamental Research Funds for the Central Universities (No. 2082006). Appendix Figures with essential colour discrimination. Certain figures in this article, particularly Figs. 2e4, 7 and 8 and Scheme 1 are difficult to interpret in black and white. The full colour image can be found in the on-line version, at doi:10.1016/j.biomaterials.2010.10.015.
References [1] Gershon D. Microarray technology: an array of opportunities. Nature 2002;416:885e91. [2] Fodor SP, Rava RP, Huang XC, Pease AC, Holmes CP, Adams CL. Multiplexed biochemical assays with biological chips. Nature 1993;364:555e6. [3] MacBeath G, Schreiber SL. Printing proteins as microarrays for highthroughput function determination. Science 2000;289:1760e3. [4] Trau M, Battersby BJ. Novel colloidal materials for high-throughput screening applications in drug discovery and genomics. Adv Mater 2001;13:975e9. [5] Han MY, Gao XH, Su JZ, Nie SM. Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules. Nat Biotechnol 2001;19:631e5. [6] Gao XH, Nie SM. Doping mesoporous materials with multicolor quantum dots. J Phys Chem B 2003;107:11575e8. [7] Gao XH, Nie SM. Quantum dot-encoded mesoporous beads with high brightness and uniformity: rapid readout using flow cytometry. Anal Chem 2004;76:2406e10. [8] Wilson R, Spiller DG, Prior IA, Veltkamp KJ, Hutchinson A. A simple method for preparing spectrally encoded magnetic beads for multiplexed detection. ACS Nano 2007;1:487e93. [9] Li J, Zhao XW, Zhao YJ, Gu ZZ. Quantum-dot-coated encoded silica colloidal crystals beads for multiplex coding. Chem Commun; 2009:2329e31. [10] Sukhanova A, Susha AS, Bek A, Mayilo S, Rogach AL, Feldmann J, et al. Nanocrystal-encoded fluorescent microbeads for proteomics: antibody profiling and diagnostics of autoimmune diseases. Nano Lett 2007;7:2322e7. [11] Rauf S, Glidle A, Cooper JM. Production of quantum dot barcodes using biological self-assembly. Adv Mater 2009;21:1e5. [12] Fournier-Bidoz S, Jennings TL, Klostranec JM, Fung W, Rhee A, Li D, et al. Facile and rapid one-step mass preparation of quantum-dot barcodes. Angew. Chem. Int. Ed 2008;47:5577e81. [13] Pamme N, Manz A. On-chip free-flow magnetophoresis: continuous flow separation of magnetic particles and agglomerates. Anal Chem 2004;76:7250e6. [14] Adams JD, Kim U, Soh HT. Multitarget magnetic activated cell sorter. Proc Natl Acad Sci U S A 2008;105:18165e70. [15] Xie HY, Zuo C, Liu Y, Zhang ZL, Pang DW, Li XL, et al. Cell-targeting multifunctional nanospheres with both fluorescence and magnetism. Small 2005;1:506e9. [16] Wang GP, Song EQ, Xie HY, Zhang ZL, Tian ZQ, Zuo C, et al. Biofunctionalization of fluorescent-magnetic-bifunctional nanospheres and their applications. Chem Commun; 2005:4276e8. [17] Xie HY, Xie M, Zhang ZL, Long YM, Liu X, Tang ML, et al. Wheat germ agglutinin-modified trifunctional nanospheres for cell recognition. Bioconjug Chem 2007;18:1749e55. [18] Song EQ, Wang GP, Xie HY, Zhang ZL, Hu J, Peng J, et al. Visual recognition and efficient isolation of apoptotic cells with fluorescent-magnetic-biotargeting multifunctional nanospheres. Clin Chem 2007;53:2177e85. [19] Xie M, Hu J, Long YM, Zhang ZL, Xie HY, Pang DW. Lectin-modified trifunctional nanobiosensors for mapping cell surface glycoconjugates. Biosens Bioelectron 2009;24:1311e7. [20] Sathe TR, Agrawal A, Nie SM. Mesoporous silica beads embedded with semiconductor quantum dots and iron oxide nanocrystals: dual-function microcarriers for optical encoding and magnetic separation. Anal Chem 2006;78:5627e32.
