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Talanta

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Quantum dots encoded Au coated polystyrene bead arranged micro-channel for multiplex arrays

Yuan-Cheng Cao a,b,1, Zhan Wang c,1, Runyu Yang a,1, Linling Zou a,b,n, Zhen Zhou a, Tie Mi d, Hong Shi a a Key Laboratory of Optoelectronic Chemical Materials and Devices of Ministry of Education, Jianghan University, Wuhan 430056, P. R. China b Flexible Display Materials and Technology Co-Innovation Centre of Hubei Province, Jianghan University, Wuhan 430056, P. R. China c Department of Chemical and Environmental Engineering, Wuhan Polytechnic University, Wuhan, Hubei Province, 430023, P. R. China d Hubei Key Laboratory of Industrial Fume and Dust Pollution Control, Jianghan University, Wuhan 430056, P. R. China article info abstract

Article history: This paper describes a promising micro-channel multiplex immunoassay method based on the quantum Received 24 March 2015 dots encoded beads which requires micro-volume sample. Briefly, Au nanoparticles coated polystyrene Received in revised form (PS) beads were prepared and Quantum dots (QDs) were employed to encode 4 types of the PS beads by 5 June 2015 different emission wavelength QDs and various intensities. Different coding types of the beads were Accepted 13 June 2015 immobilized with different antibodies on the surface and BSA was used to block the unsatisfied sites. The antibody linked beads were then arranged in the 150 mm diameter optical capillary where the specific Keywords: reactions took place before the detections. Results showed that the antibody on the Au coated surface Quantum dots maintains the bioactivity for the immunoreactions. Using this system, the fluorescent intensity was Multiplex linear with analyte concentration in the range of 1 10 7–1 10 5 mg/mL (RSDo5%, 4 repeats) and the Capillary immunoassays lower detection limit reached 5 10 8 mg/mL. It was proved to be a promising approach for the future Biochips Fluorescent analysis miniaturization analytical devices. & 2015 Elsevier B.V. All rights reserved.

1. Introduction molecules are physically or chemically attached on the separated wells of a plate at a particular location in an array. It is the exact Recent trends in analytical chemistry have shifted toward the location information (x, y coordinates) on the microtitre plate or miniaturized multiplex analytical systems that integrate sample microarray which allows the identification of the molecule that is preparation and measurement steps into micro-fabricated fluidic analyzed at that location. This is also called positional (or spatial) devices [1–7]. Numbers of strategies aimed at these applications encoding/decoding method [10–14]; (b), the reactions are carried “ have been created, and the promising tools are the multiplex out on individual encoded microcarrier (polymer, glass beads or ” technologies which allow multiple discrete assays to be carried other particles) with a particular probe bound to its surface – out simultaneously using micro-volume samples [8 11]. Multi- [8,9,14]. This method allows encoded microcarriers to be mixed plexing and miniaturization become pervasive themes in analy- and subjected to an analysis simultaneously. Those microcarriers tical chemistry where large numbers of candidate molecules and show a favorable reaction between the attached probe and the target molecules are involved. Thus, the requirements to measure target [8–10]. The former strategy (a) exploits the positional en- ever-increasing numbers of species from smaller volumes have led coding to carry out the analyses in parallel, but it suffers from slow to innovative devices for sample manipulation and simultaneous diffusion and limitations of analyte in the solid–liquid surface capabilities [10,11]. [14–16]. While in the later strategy (b), distinctive micro-carriers One of the most important challenges in developing multiplex such as micro beads, are accomplished by incorporation molecules assays is the necessity to track and decode the individual reaction. with better contact surface than that of the planar formats [4,9,17– Generally, there are two main strategies to achieve this: (a) the 20]. Bead arrays circumvent the shortcomings of planar platforms for analytical developments. Therefore, micro-bead based arrays n Corresponding author at: Key Laboratory of Optoelectronic Chemical Materials make another important step in the direction of the universal and Devices of Ministry of Education, Jianghan University, Wuhan 430056, China. chemical and biological detection platform. E-mail addresses: [email protected] (Y.-C. Cao), [email protected] (Z. Wang). Semiconductor quantum dots (QDs) are one of the promising 1 These authors contributed equally to this work. optical encoding materials that have the novel properties such as http://dx.doi.org/10.1016/j.talanta.2015.06.030 0039-9140/& 2015 Elsevier B.V. All rights reserved.

Please cite this article as: Y.-C. Cao, et al., Talanta (2015), http://dx.doi.org/10.1016/j.talanta.2015.06.030i 2 Y.-C. Cao et al. / Talanta ∎ (∎∎∎∎) ∎∎∎–∎∎∎ narrow FWHM (full width at half maxima), tunable emissions, and Au@PS beads. This was completed in 2 mL of chloroform, which same excitation for different emission QDs. Therefore, QDs are had been added to 0.25 g Au@PS beads and 2 mL of QDs solution. widely used as encoding materials and this area is becoming the The mixture was stirred until no fluorescence signal observed in attractive research field [3,18–22]. Some researchers found that the solution according to the fluorescent tests. The doping process the Au nanoparticles on the matrix surface can effectively improve was completed within 2 h at room temperature. After the doping, the fluorescent properties [20,21]. the encoded beads were isolated by filter and washed for several Herein we describe a new approach which is suitable for the hours with ethanol to obtain the stable QDs encoded Au@PS beads rapid detection of immunoassay using a bead-based capillary. Each (QDs–Au@PS). micro-bead is encoded with a unique bar code before the antibody The QDs stability test for the resultant QDs–Au@PS sample was was attached. This “optical bar code” is simply a combination of carried out by the fluorescent tests. Briefly, the QDs–Au@PS beads

QDs with different emission wavelengths and intensities which (0.2 g) were dispersed in 2 mL deionized H2O in the tube and kept allows each bead to be independently identified. Then different shaking for 48 h. The wash solution was then used for the fluor- beads are arranged inside the capillary to assembly a set of probe escent test to determine if the QDs were leaked out from the array. The potential advantages of this bead-arrayed capillary Au@PS beads. analytical strategy are including low cost, shorter analysis time, higher sensitivity, portability and simplicity in operation, com- 2.4. Immobilization of probes to QDs encoded Au@PS beads pared to conventional microscopic instrumentation. The immobilization of monoclonal IgG antibodies was accom- plished by the immersion of the QDs–Au@PS beads into a 50 mM 2. Experiment section phosphate buffer (PBS) at pH 6.0 with the addition of 1% (v/v) Tween-80 and 1 mg/mL antibody. Tween-80 was incorporated 2.1. Chemical and reagents into the PBS solution to minimize any free IgG proteins on the surface. Then 5% Bovine Serum Albumin (BSA) solution (1%) was Tri-n-octylphosphine oxide (TOPO, 90%), CdO (99.99%), sele- added to block the unoccupied sites. After the blocking step, the nium powder (99.99%) were ordered from Aldrich. Sodium citrate mixture was centrifuged and washed to get the beads. The re- (99%), HAuCl (98%) were purchased from China National Phar- 4 sultant beads (antibody linked QDs–Au@PS) were divided into maceutical Group (Shanghai, China). Polystyrene (PS) beads several portions. Some of the beads were used for bioactivity tests (107710 mm, 1% cross-linked) were obtained from Aldrich. The and some were pumped into the capillaries for immunoassays. hollow fiber capillary (150 mm of the inner diameter) was snipped The optical capillary was first inserted into a rubber ring (out into short pieces of 50 mm in length. Rabbit IgG, FITC-labeled diameter 160 mm and inner diameter 80 mm) at one end and then goat-anti-Rabbit IgG and FITC-labeled goat-anti human IgG anti- the PBS buffer with antibody linked QDs–Au@PS beads were body (affinity purified) were provided by Beijing Zhongshan pumped into the capillary through the open end to the rubber ring Golden Bridge Biotechnology Co., Ltd. (China). All other reagents end. The beads were then stopped by the rubber ring one by one required for use in the coupling, washing or hybridization buffer to make the beads array (see Supplementary data S1). were of analytical grade and were used as received.

2.5. The capillary based immunoassays 2.2. Preparation of colloidal Au and Au coated PS beads

Tests for bioactivity of the immobilized IgG were conducted at The Au nanoparticles were prepared according to the Ref. [21]. “ ” Briefly, 1 mL of a 1% (w/v) HAuCl solution was added to 40 mL of the goat anti-rabbit IgG contained sample in binding buffer : 4 – triply distilled water and heated to boil while stirring, then 1.5 mL 100 mM Tris HCl (pH 7.6), 100 mM NaCl, 15 mM magnesium of a 1% (w/v) trisodium citrate solution was added drop-by-drop chloride, and 1% (v/v) Tween 80. The tests were conducted using fl while kept stirring and the resulting mixture was boiled for an- the uorescein isothiocyanate (FITC) labeled goat anti-rabbit an- – “ other 15 min. Then the Au colloids were cooled down in room tibody. QDs Au@PS-rabbit IgG beads were added into the binding ” temperature. Before the experiments, the glassware was thor- buffer above mentioned which had added the FITC-labeled goat anti-rabbit IgG sample for immunoreaction in Eppendorf tube. oughly cleaned with the aqua regia (HNO3:HCl¼1:3) and deio- Then the tube was shaked and incubated in room temperature for nized H2O, and then oven-dried at 100–110 °C for 3 h. Au coated PS beads were prepared by the deposition of Au 10 min. Then the IgG immobilized beads were washed with the fl nanoparticles on the surface of the beads. 5 mL of the Au colloids PBS buffer for 3 times before the uorescence detection. was put into the 10 mL centrifugal tube and 0.025 g PS beads The detection experiments were carried out using the similar procedures as the bioactivity tests. The beads for each encoding (surface grafted with –NH2 terminal according to Ref. [22] were added into the tube. Then the tube was shaked for 24 h before the type in the arrays were used with 4 repeats inside the capillary. tube was centrifuged to get the beads, and the beads were washed by water and ethanol for several times in turn, until there were no 2.6. Characterization Au nanoparticles in the washed solution by UV tests. Then the Au coated PS beads (Au@PS) were dried in room temperature. The fluorescence characteristics of QDs were determined by Luminescence Spectrometer (LS-55, Perkin-Elmer, USA). The 2.3. Preparation of ZnS-capped CdSe QDs and QDs encoded Au@PS fluorescence spectrum of the beads was taken from the surface of beads the beads using UV–vis fiber optic spectrometer (HR2000, Ocean Optics, USA). Core-shell QDs (ZnS-capped CdSe) were synthesized according The beads for Scanning Electron Microscope (SEM, S-3000N, to Refs. [18,19]. The synthesized QDs were coated with a layer of Hitachi, Japan) studies were dried in room temperature before the tri-n-octylphosphine oxide (TOPO), which was used as a high test. temperature coordinating solvent. Optical and fluorescence images were taken by digital camera Two-color encoded beads with various ratios of QDs were (A80, Canon, Japan) from the Inverted Fluorescence Microscope completed by doping a controlled amount of QDs into porous (IX71, Olympus) with mercury lamp (λ¼400–440 nm).

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Fig. 2. Stability test of QDs immobilization in Au@PS beads in chloroform (a) and PBE buffer (b). The data were determined by accumulated intensity of QDs leaked for the QDs–Au@PS beads and each data point repeated three times.

fluctuations were observed in the test conditions. These results indicate that Au@PS beads are the promising encoding carriers for the multiplex analysis. The QDs stability tests were carried out using the encoded QDs–Au@PS beads. And this coding stability determines the code precision and identification veracity. The results are shown in Fig. 2. In the experiments, organic solvent chloroform and water solvent PBS buffer were used. From the results we can see that the amount of QDs in the washing solution from both chloroform and PBS buffer was increased rapidly in first several hours and then became steady. For the PBS buffer, the QDs encoded beads became steady in 4 h but for the chloroform solvent, this took 16 h to reach its stabilization. And also more QDs were leaked in the chloroform solvent than the PBS buffer. This might be caused by the swelling Fig. 1. (a) The spectra of QDs in solution. (a) λem¼576 nm; (b) λ¼628 nm; (c) λ¼652 nm. Excited at 365 nm. (b) Encoding signals of arrayed beads in capillary for of the PS bead in chloroform. As most of the bioanalysis are carried multiplex analysis. The encoding is comprised of wavelength and intensity; for A out in the aqueous phase, the QDs encoding is stable after the λ ¼ and B, the em 576 nm and 628 nm intensity ratio is 1:1 (curves1,2) and 1:2 washing of the encoding preparation. (curves3,4) respectively; for C and D, the λ ¼576 nm and 652 nm intensity ratio is em The immobilization of the antibody on the QDs–Au@PS beads 2:1 (curves5,6) and 1:1 (curves7,8) respectively. was achieved by the absorbance of the IgG onto the Au coatings and BSA blocking of the unoccupied sites. IgG protein has amino 3. Results and discussions acids and –SH groups which can bind with the Au nanoparticles on the PS surface [13,15,21]. BSA blocking of the unsatisfied sites We synthesized 3 emission wavelength QDs which are soluble aimed to eliminate the unspecific binds in the detections. Four in organic solvent and the spectra are shown in Fig. 1(a). The encoded QDs–Au@PS beads were applied to attach four different spectra show that the FWHM was 25 nm. Au coated Polystyrene antibodies to show the feasibility of this strategy: A type (rabbit Beads (PS beads) were prepared by self-assembly [21]. Compared IgG), B type (human IgG), C type (goat IgG) and D type (mouse to the PS beads before the coating, the surface of the coated PS IgG). beads was covered by the Au particles and thus formed porous In order to test the antibody bioactivity on the QDs–Au@PS surface structure (Supplementary data S2). The QDs doping takes beads, Type A bead that immobilized rabbit IgG was applied to advantages of the porous structure during the coding process. The immunoreact with the FITC labeled goat-anti-rabbit IgG and FITC- resultant core/shell PS beads not only have the biocompatibility goat-anti-human IgG, respectively. The results showed that after from the coated Au nanoparticles to immobilize the antibodies on the reaction with the FITC-goat-anti-rabbit IgG, FITC signal was the surface, but also can be used as the QDs encoding carriers for observed and the intensity was related to the concentration of the the immunoassays. target analytes (see Supplementary data S3). While for the FITC- The coding was achieved by the doping of various amounts of goat-anti-human IgG sample, no FITC signal was detected al- the different wavelength QDs into the Au coated PS beads (QDs– though higher concentration of the goat-anti-human IgG in the Au@PS). As the fluorescent materials, QDs encoding includes wa- test samples was applied. These results indicate that the IgG im- velength and intensity. Emission wavelength (576 nm, 628 nm and mobilized on the QDs–Au@PS beads maintains the im- 652 nm) QDs were employed for doping the Au@PS beads in var- munoactivity to achieve the specific reaction. ious intensities and the resultant encoded beads are shown in After the bioactivity tests, these antibodies attached Fig. 1(b). From the encoding spectra we can see that the encoding QDs–Au@PS beads were arranged into the capillary to make the signals can be easily distinguished by the intensities and wave- four encoded PS beads capillary array in the 150 mm hollow optical lengths. These encodings are steady and stable, no overlaps or fiber (Fig. 3). From the bright field photo of the capillary we can

Please cite this article as: Y.-C. Cao, et al., Talanta (2015), http://dx.doi.org/10.1016/j.talanta.2015.06.030i 4 Y.-C. Cao et al. / Talanta ∎ (∎∎∎∎) ∎∎∎–∎∎∎

Fig. 5. Detected spectra of arrayed beads in capillary after the immunoreaction. The concentration of rabbit-anti-mouse IgG was 0.005 mg/mL, and the FITC labeled goat-anti-rabbit IgG was excessive.

reaction process, the bead arrays are scanned by the detector to collect and analyse the spectra encoding signal and reporter in- tensities for the decoding and qualitative measurements of the analytes in the samples. fi fl – Fig. 3. Bright eld (A) and uorescence (B) image of a probe immobilized QDs In order to study the feasibility of this detection system, rabbit Au@PS bead capillary. anti-mouse IgG used as analyte in the sample was applied to test see there was 50 mm gap between the bead and the inner wall the QDs encoded bead capillary arrays and the FITC labeled goat (Fig. 3A) and this gap is for the diffusion and flow of the tested anti-rabbit IgG was used as reporter for the analyte, and the re- samples (Fig. 3B). In the analysis, one end of the capillary was sultant spectra are shown in Fig. 5. From the spectra we can see immersed in the sample solution and the solution went up in the that none of the A, B, C type beads was detected with FITC fluor- capillary by the capillary action. Then the target analytes were escence, but for the type D beads at 520 nm there was obvious captured by the probes in the encoded beads by the specific re- FITC signal which was contributed from the FITC-goat anti-rabbit actions. After the PBS buffer washings, FITC labeled second anti- IgG corresponding to the target analyte rabbit anti-mouse IgG. This body was applied to form the “sandwich” immunoassay reactions result indicates that the QDs encoded beads in the capillary array (see Supplementary data S4). have the good specific immunoreactions in the immunoassays. The encoding beads array detection system is shown in Fig. 4. Further tests showed that in a certain range, the intensity of the fl fi This system includes uorescent microscopy, ber optic spectro- fluorescence is linear with the target analyte concentration (Fig. 6). meter and data collect/analysis computer. This system uses PS From the results we can see that when excess FITC-goat-anti- beads as the probe carriers to detect the target molecular, not the rabbit IgG was applied, the detectable analyte rabbit-anti-mouse traditional planar biochips. In the individual reaction, the target is IgG concentration was linear with the intensity in the range of captured by the unique beads on the bead surface. After the 1 10 7–1 10 5 mg/mL (RSDo5%, 4 repeats) with the linear regression

Fiber Optic Spectrometer

Spectra analysis

Fluorescence for detection Emission filter Excitation filter Dichromatic mirror

Mercury burner

Spectra Scan

Probe 1 Probe2 Probe3 ...... Probe n Fig. 6. The correlation of target concentration and intensity. The target was rabbit- Fig. 4. Scheme of bead arrays in a capillary system for the multiplex analysis. anti-mouse IgG and the fluorescence was detected from the D type bead.

Please cite this article as: Y.-C. Cao, et al., Talanta (2015), http://dx.doi.org/10.1016/j.talanta.2015.06.030i Y.-C. Cao et al. / Talanta ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 5

YR[]=−In 7.74 + 2832 ×[] Con.( == 99.92%, SD 0.252) () 1 Development Program of China (863 Program: 2015AA033406). And Prof. Tie Mi thanks the funds from the Science and Technology 8 When the analyte concentration was as low as 5 10 mg/mL, Project of Wuhan City (2013061001010481), the Natural Science there was detectable FITC signal. These results showed that the Foundation of China (U1261204), Hubei Province Innovative Young QDs encoded bead in capillary format possesses stable perfor- Research Team in Universities (T201420). mance and high sensitivity to the analytes. This is a promising approach for the efficient and economic immunoassay which re- quires micro-volume samples and this integration of encoded bead Appendix A. Supplementary material with the capillary extends the fluorescent analysis and suspended chips in the chemical and biological analysis. Supplementary data associated with this article can be found in the online version at doi:10.1016/j.bios.2014.05.063.

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Please cite this article as: Y.-C. Cao, et al., Talanta (2015), http://dx.doi.org/10.1016/j.talanta.2015.06.030i