Rapid and Sensitive Biomolecular Screening with Encoded

Apr 1, 2010 - E-mail: [email protected]. ... The suspension array, which used these macroporous hydrogel photonic beads as coding elements, showed ...
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Rapid and Sensitive Biomolecular Screening with Encoded Macroporous Hydrogel Photonic Beads Yuanjin Zhao,† Xiangwei Zhao,† Baocheng Tang,† Wenyu Xu,† and Zhongze Gu*,†,‡ †

State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China, and ‡Suzhou Key Laboratory of Environment and Biosafety, Suzhou 215123, China Received January 28, 2010. Revised Manuscript Received March 22, 2010 We present a new method to prepare inverse opaline photonic beads with good spherical shape and superior optical performance by simply introducing an interfacial tension system into a template replication method. When the scaffolds of these beads were composed of poly(ethylene glycol) diacrylate hydrogel, they could provide a homogeneous water surrounding, which remedied many shortcomings of biomolecular microcarriers introduced by the presence of the solid surface of them. The suspension array, which used these macroporous hydrogel photonic beads as coding elements, showed obvious advantages in multiplexed capability, rapid biomolecular screening (within 12 min), and highly sensitive detection (with limit of detection of ∼10-12 M).

Introduction Rapid and multiplexed molecular screening with high sensitivity and specificity is of great importance in gene profiling, clinical diagnostics, and environmental monitoring.1-3 A key requirement for multiplexing is the identification of different binding events in parallel. In this context, suspension arrays,4,5 in which probes are attached to the surface of microparticles, have revolutionized DNA expression profiling and show great promise for proteomics and DNA sequencing.6-8 Compared with conventional microarrays on a plate, the suspension arrays offer greater flexibility in the preparation of new assays and higher diffusional flux of analytes due to radial diffusion and are less expensive to produce.9 Among various suspension arrays, those arrays with spectrum-encoded elements, such as fluorescent dyes and quantum dots,10-14 are well used due to their simplicity in both encoding and detection. Most of the commercialized products are based on this approach. However, there are several disadvantages of using fluorescence dyes or quantum dots as means of encoding elements, including the photobleaching during storage and the potential interference of encoding fluorescence with analyte-detection fluorescence. Recently, photonic beads appeal themselves as a new type of spectrum-encoding carrier, whose *To whom correspondence should be addressed. E-mail: [email protected].

(1) Broude, N. E. Trends Biotechnol. 2002, 20, 249. (2) Heller, M. J. Annu. Rev. Biomed. Eng. 2002, 4, 129. (3) Li, M. Z.; He, F.; Liao, Q.; Liu, J.; Xu, L.; Jiang, L.; Song, Y. L.; Wang, S.; Zhu, D. B. Angew. Chem., Int. Ed. 2008, 47, 7258. (4) Nolan, J. P.; Sklar, L. A. Trends Biotechnol. 2002, 20, 9. (5) Wilson, R.; Cossins, A. R.; Spiller, D. G. Angew. Chem., Int. Ed. 2006, 45, 6104. (6) Meza, M. B. Drug Discovery Today 2000, 1, 38. (7) Walt, D. R. Science 2000, 287, 451. (8) Nam, J. M.; Thaxton, C. S.; Mirkin, C. A. Science 2003, 301, 1884. (9) He, B.; Son, S. J.; Lee, S. B. Anal. Chem. 2007, 79, 5257. (10) Battersby, B. J.; Bryant, D.; Meutermans, W.; Matthews, D.; Smythe, M. L.; Trau, M. J. Am. Chem. Soc. 2000, 122, 2138. (11) Han, M. Y.; Gao, X. H.; Su, J. Z.; Nie, S. M. Nat. Biotechnol. 2001, 19, 631. (12) Wang, D. Y.; Rogach, A. L.; Caruso, F. Nano Lett. 2002, 2, 857. (13) Kuang, M.; Wang, D. Y.; Bao, H. B.; Gao, M. Y.; Mohwald, H.; Jiang, M. Adv. Mater. 2005, 17, 267. (14) Zhao, Y. J.; Zhao, X. W.; Tang, B. C.; Xu, W. Y.; Li, J.; Hu, J.; Gu, Z. Z. Adv. Funct. Mater. 2010, 20, 976. (15) Cunin, F.; Schmedake, T. A.; Link, J. R.; Li, Y. Y.; Koh, J.; Bhatia, S. N.; Sailor, M. J. Nat. Mater. 2002, 1, 39.

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code is the characteristic reflection peak originated from the stopband.15-18 As the peak position is based on their periodical structure, the code is very stable and the fluorescent background is relatively low. These properties make the photonic beads suitable for highly sensitive detection. Although suspension arrays show many significant advantages, there remain certain drawbacks introduced by the presence of the solid surface of microparticles, such as limited binding kinetics, reduced activity of surface-bound biomolecules, and insufficient control of the process of surface adsorption.19 In this point of view, hydrogels, which provide a rather homogeneous water surrounding, have been regarded as an ideal alternative substrate or platform for biomolecular screening. As these threedimensional (3D) hydrogels show high capacity for biomolecule immobilization, greater probability of interacting with the target ligand, high LOD, S/N, and sensitivity,20-22 they have been commercialized by some companies in microarrays. However, there is little research on the suspension array using hydrogel microparticles as encoding carriers,23,24 and development of new kinds of encoded hydrogel microparticles is anticipated. In this paper, we designed a new type of suspension array for biomolecular screening by using macroporous hydrogel photonic beads as coding elements. Up to now, a number of methods have been developed for the fabrication of ordered macroporous materials, such as cocrystallization and template replication. However, it is still a challenge to prepare the macroporous photonic beads with perfect spherical shape and long-range (16) Zhao, Y. J.; Zhao, X. W.; Sun, C.; Li, J.; Zhu, R.; Gu., Z. Z. Anal. Chem. 2008, 80, 1598. (17) Meade, S. O.; Chen, M. Y.; Sailor, M. J.; Miskelly, G. M. Anal. Chem. 2009, 81, 2618. (18) Hu, J.; Zhao, X. W.; Zhao, Y. J.; Li, J.; Xu, W. Y.; Gu., Z. Z. J. Mater. Chem. 2009, 19, 5730. (19) Krishnamoorthy, S.; Himmelhaus, M. Adv. Mater. 2008, 20, 2782. (20) Ali, M. F.; Kirby, R.; Goodey, A. P.; Rodriguez, M. D.; Ellington, A. D.; Neikirk, D. P.; McDevitt, J. T. Anal. Chem. 2003, 75, 4732. (21) Martin, B. D.; Soto, C. M.; Taitt, C.; Charles, P. T. Biosens. Bioelectron. 2008, 99, 1241. (22) Charles, P. T.; Taitt, C. R.; Goldman, E. R.; Rangasammy, J. G.; Stenger, D. A. Langmuir 2004, 20, 270. (23) Pregibon, D. C.; Toner, M.; Doyle, P. S. Science 2007, 315, 1393. (24) Pregibon, D. C.; Doyle, P. S. Anal. Chem. 2009, 81, 4873.

Published on Web 04/01/2010

DOI: 10.1021/la100939d

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ordering pores. Though the method of co-crystallization could fabricate the beads, the proportion of template particles and nanoparticles should be controlled rigorously which was difficult to carry out.25,26 A conventional template replication method can produce porous beads with long-range order,27,28 but it needs careful separation of the beads from the template films. Herein, to meet the demand, we developed a versatile method for the fabrication of macroporous photonic beads by simply introducing an interfacial tension system into the template replication method. When the scaffolds of these beads were composed of poly(ethylene glycol) diacrylate hydrogel, they provided a homogeneous water surrounding for bioassay. It was demonstrated that our suspension array showed obvious advantages in rapid biomolecular screening and highly sensitive detection.

Experimental Section Materials. Monodisperse silica nanoparticles with diameters ranging from 186 to 300 nm were synthesized by the St€ ober method. Poly(tetrafluoroethylene) (PTFE) pipes with inner diameters of 500 μm and T-junction (P-727) were purchased from Upchurch Scientific, Oak Harbor, WA. Poly(dimethylsiloxane) (KF-96 10 cSt) was acquired from Shin-Etsu Chemical, Japan. Silicon oil was purchased from Yunuo Chemicals Ltd., China. Poly(ethylene glycol) diacrylates (PEG-da) with weight-average molecular weights of 700 and 2-hydroxy-2-methylpropiophenone photoinitiator were purchased from Aldrich. Oligonucleotide probes (probe 1, 50 -NH2-(CH2)6-TGA TCG CGG TGT CAG TTC TTT-30 ; probe 2, 50 -NH2-(CH2)6-GTG GAA TTG AGC AGC GTT GGT-30 ) and 7-amino-4-methylcoumarin (AMC) tagged oligonucleotide targets (target 1, 50 -(AMCA)-AAA GAA CTG ACA CCG CGA TCA-30 ; target 2, 50 -(AMCA)ACC AAC GCT GCT CAA TTC CAC-30 ) were obtained from TaKaRa Biotechnology (Dalian) Co., Ltd. TE buffer (10; 100 mM Tris-HCl, 10 mM EDTA, pH 8.0), wash buffer (0.2 SSC 0.2% SDS), and hybridization buffer (750 mM NaCl, 150 mM sodium citrate, pH 7.4) were all self-prepared. Deionized water was used for all experiments. Instrumentation. The microfluidic device used for template silica colloidal crystal bead (SCCB) generation was homemade.16 Photographs of macroporous hydrogel photonic beads were taken by using an optical microscope (OLYMPUS BX51) equipped with a CCD camera (MediaCybernetics Evolution MP 5.0). Reflection spectra of the beads were recorded by using an optical microscope equipped with a fiber optic spectrometer (Ocean Optics, USB2000-FLG). Broadband excitation in the near-UV range (330-385 nm) was provided by a 100 W mercury lamp. A long-pass dichroic filter (DM 400, Chroma Technologies, Brattleboro, VT) was used to reject the scattered light and to pass the Stokes-shifted fluorescence signals. Fluorescence spectra of the beads were recorded by using a microscope objective equipped with a fiber optic spectrometer (Ocean Optics, QE65000).

Preparation of Macroporous Hydrogel Photonic Beads. The monodisperse and size-controlled SCCBs were fabricated by using the microfluidic device16 and were calcined to 800 °C for 3 h before use. The macroporous hydrogel photonic beads were replicated from the void of the SCCBs. To make sure the pregel solution can fill the void fully, the SCCBs were first treated with piranha solution (30% hydrogen peroxide and 70% sulfuric acid) for 6 h. After washing with water and drying by nitrogen flow, the SCCBs were immersed in the pregel solution (39% PEG, 1% initiator, 2% acrylic acid, 58% H2O) for 1 h. Then the pregel (25) Moon, J. H.; Yi, G. R.; Yang, S. M.; Pine, D. J.; Park, S. B. Adv. Mater. 2004, 16, 605. (26) Zhao, Y. J.; Zhao, X. W.; Hu, J.; Xu, M.; Zhao, W. J.; Sun, L. G.; Zhu, C.; Xu, H.; Gu, Z. Z. Adv. Mater. 2009, 21, 569. (27) Yi, G. R.; Moon, J. H.; Yang, S. M. Chem. Mater. 2001, 13, 2613. (28) Zhao, Y. J.; Zhao, X. W.; Hu, J.; Li, J.; Xu, W. Y.; Gu, Z. Z. Angew. Chem., Int. Ed. 2009, 48, 7350.

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solution containing the immersed SCCBs was dispersed into silicon oil with continuous stirring (∼400 rpm, 30 min). Next the beads were exposed to UV light for the polymerization of the pregel solution left in the voids of the opal structure. About 20 min later, the hybrid beads were filtered from the oil and washed with hexane, ethanol, and water. Finally, the macroporous hydrogel photonic beads were obtained after removing the template SCCBs with hydrofluoric acid (1-2%). Biomolecular Screening. For the biomolecular screening, oligonucleotide probes (50 μM) were immobilized to the macroporous hydrogel photonic beads in the presence of coupling reagents (EDC 5 mM, NHS 0.05 M) at room temperature for 3 h and 4 °C overnight. After being washed with TE buffer and HB buffer, the macroporous hydrogel photonic beads were used as the carriers for DNA hybridization in test tubes. Different concentrations of targets were used to incubate the beads (1 μL per bead). During the hybridization process, the tubes were shaken at 37 °C. The unbound oligonucleotide targets were washed away by electrophoresis (80 V, 10 min). The fluorescence spectra of the beads were measured after washing. For comparison, the SCCBs and glass beads with the same size as the macroporous hydrogel photonic beads were treated with 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde for 4 h, respectively. Then oligonucleotide probes were immobilized to them by chemical bonding under optimized conditions.16 The number of replicates at any concentration was 5. The detection limit was calculated from the zero calibrator plus three times the standard deviation.

Results and Discussion Fabrication of Macroporous Hydrogel Photonic Beads. In this study, the silica colloidal crystal beads (SCCBs)16 were used as a sacrificial template to create macroporous hydrogel photonic beads with 3D, highly ordered, inverse opal structures (scheme of the bead fabrication in Supporting Information Figure S1). The SCCBs were first immersed in the pregel solution. After the pregel solution went through the void spaces between the silica nanoparticles of the SCCBs by capillary force, the mixture was dispersed into silicone oil with continuous stirring. During this process, the pregel solution on the bead surface could be broken into small droplets by the oil flows, while the solution in the void spaces remained in the SCCBs due to the capillary force. When most of the pregel solution on the bead surface was dispersed, the beads were exposed to UV light for the polymerization of the pregel solution left in the void spaces of the opal structure. After the SCCB template of the hybrid beads was etched by hydrofluoric acid, the macroporous hydrogel photonic beads could be successfully produced. This method can also be used to prepare other kinds of inverse opaline photonic beads by choosing appropriate templates and interfacial systems. To meet the demand of sensitive biomolecular screening, poly(ethylene glycol) (PEG) was used as the element of macroporous hydrogel photonic beads due to its bioinert property and eliminable nonspecific binding of proteins and oligonucleotides. We prepared the hydrogel beads with different reflection peaks by using different concentrations of PEG (Supporting Information Figure S2). It was found that although the beads fabricated with a high concentration of PEG had better mechanical strength, they showed obvious swelling properties which were disadvantageous to maintaining the long-range ordering of pores and stable reflection peak position. Generally, the hydrogels composed of less than 40% PEG had negligible swelling and exhibited sufficient diffusion of oligomers in and out of them.29 Herein, we used (29) Meiring, J. E.; Schmid, M. J.; Grayson, S. M.; Rathsack, B. M.; Johnson, D. M.; Kirby, R.; Kannappan, R.; Manthiram, K.; Hsia, B.; Hogan, Z. L.; Ellington, A. D.; Pishko, M. V.; Willson, C. G. Chem. Mater. 2004, 16, 5574.

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Figure 1. SEM images of the surface of (a) template SCCBs and (b) PEG inverse opaline photonic beads.

Figure 2. (a) Effects of incubation time on fluorescence intensities of microcarriers in 1 μM target DNA 1 solution. (b) Relationship between the fluorescence intensity and the concentrations of fluorescence tagged target DNA1 used for hybridization. The number of replicates at any concentration was five. Error bars represent standard deviations.

40% PEG to fabricate all the macroporous hydrogel photonic beads for biomolecular screening. Figure 1 shows the surface scanning electron microscopy (SEM) images of the template SCCBs and the macroporous hydrogel photonic beads. It can be seen that the macropores on the surface of the hydrogel beads formed the same hexagonal alignment as the silica nanoparticles on the SCCBs. In addition, the macropores were interconnected and extended to the inside of the inverse opaline building-block PEG beads. This structure not only can provide larger surface area and more probe sites, but also can offer easier accessibility for the target DNA to the probe DNA. Performance of the Macroporous Hydrogel Photonic Beads. To demonstrate the potential advantages of using the macroporous hydrogel photonic beads as carriers in biomolecular screening, they were compared with SCCBs and glass beads of the same size, which were with porous structures and solid surfaces. Figure 2 shows the relationship between the fluorescence intensities and hybridization times, together with the dose-response curve of the three kinds of carriers. The results clearly indicate that the fluorescence intensities on the macroporous hydrogel photonic beads increased quickly and reached their maximum values in about 12 min, and their limit of detection was found to be ∼10-12 M without biotin-avidin-aided signal amplification, which exceeds about 1 order of magnitude of SCCBs and 3 orders of glass beads. The advantages of the macroporous hydrogel photonic beads are mainly ascribed to their structural properties and material characters. When using simple solid surfaces as carriers, such as glass beads, the fluorescence signals were weak and difficult to detect. After introducing porous structures to the carriers, such as SCCBs, the signals were amplified obviously. The reason is that the porous SCCBs provide a high surface-to-volume ratio (SVR), and for a given volume higher SVR means larger surface area for DNA hybridization and stronger fluorescence signal. However, it was time-consuming to finish the hybridization due to the low diffusion velocity of targets, limited reaction Langmuir 2010, 26(9), 6111–6114

kinetics, and fast sedimentation of the microcarriers caused inefficient hybridization. These shortcomings can be remedied by using PEG hydrogel as the scaffold of inverse opaline photonic beads. They not only can keep the high SVR, but also can provide a three-dimensional hydrogel grid for the probe DNA immobilization and biomolecular screening. The beads can also provide a higher volume fraction of pores (about 74%) and homogeneous water surrounding, both of which are beneficial for fast radial diffusion of targets and accelerate the reaction kinetics of DNA hybridization. In addition, different from the case of silica microcarriers, which have a large density and fast sedimentation speed, the sedimentation speed of the PEG hydrogel macroporous hydrogel photonic beads is greatly reduced. This makes them more suitable for real application. In other words, the macroporous hydrogel photonic beads were more suitable for rapid biomolecular screening and highly sensitive detection. Multiplexed Biomolecular Screening. In multiplex and high-throughput biomolecular screening, a number of individual assays need to be carried out simultaneously within the same sample. For the suspension arrays, the carriers, on which probe biomolecules such as DNA strands or proteins are immobilized to bind the target molecules, should be self-encoded. The code elements of the macroporous hydrogel photonic beads are the positions of their reflection peaks, which depend on the structural periodicity and can be estimated from Bragg’s law under normal incidence: λ ¼ 1:633dnaverage

ð1Þ

where λ is the peak wavelength, d is the center-to-center distance between two neighboring nanopores formed after etching the silica nanoparticles in PEG, and naverage is the average refractive index of the macroporous hydrogel photonic beads. As naverage is a constant under fixed PEG concentration of the beads, the peak wavelength λ mainly depends on the center-to-center distance DOI: 10.1021/la100939d

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Figure 3. Reflection spectra (left) and fluorescence microscope images (right) of the three kinds of macroporous hydrogel photonic beads in multiplexed analysis. Insets in the reflection spectra are the reflection images of their corresponding beads. For a few kinds of target assays, the probe could be distinguished by the color of the beads, while for a large number of target assays the probe DNA in the beads could be read out exactly by analyzing the Bragg peak positions.

between two neighboring nanopores. Therefore, a series of macroporous hydrogel photonic beads with different diffraction peak positions and colors could be obtained (Supporting Information Figure S3) by changing the diameters of the pores, which could be realized by using different sizes of silica nanoparticle assembled SCCB templates. For a few kinds of target assays, the probe could be distinguished by the colors of the macroporous hydrogel photonic beads. While for a large number of target assays it is difficult to distinguish the probe by the color of the photonic beads, the beads could be read out exactly by analyzing their Bragg peak positions. Because the macroporous hydrogel photonic beads’ codes originate from their periodical structure, they are very stable and do not suffer from fading, quenching, and chemical instability. In addition, because no dyes or materials related with fluorescence are introduced, the fluorescence background of the beads is very low. These properties make them suitable to be utilized as self-encoded carriers. Here, three kinds of macroporous hydrogel photonic beads with reflection peak positions at 627, 576, and 528 nm, respectively, were used to demonstrate multiplexing capabilities. They were loaded with oligonucleotide probes for DNA sequence detection. The beads with the reflection peak position at 627 nm were loaded with 20-base pair (bp) oligonucleotide probe 1 (50 -TGA TCG CGG TGT CAG TTC TTT-30 ), another at 576 nm

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with probe 2 (50 -GTG GAA TTG AGC AGC GTT GGT-30 ), and the third at 528 nm with no probe, to serve as a control. Targets were fluorescently labeled oligonucleotides with complementary sequences to the two probes. The beads were mixed together and incubated at room temperature in microwells each containing target 1 (at 10 μM), target 2 (at 10 μM), both targets (both at 5 μM), or no target. Because of the specific binding between the probe DNA and its corresponding fluorescently labeled target DNA, it was expected that the fluorescence signals would be observed only on the beads for which their corresponding targets were present. Figure 3 shows the results of multiplex DNA sequence screening. In each instance, it can be observed that the beads show uniformity with high specificity to the oligomers, exhibiting fluorescence only when the target was present. In our experiments, only one fluorescent dye was used and thus there was no interference when fluorescence occurred, which allowed us to avoid the fluorescence spectral overlap problem existing in most color-encoded particles arrays.

Conclusions The macroporous PEG photonic beads with good spherical shape and superior optical performance were successfully fabricated by simply introducing an interfacial tension system into a template replication method. When used in bioassay, the hydrogel beads could provide a homogeneous water surrounding, which may remedy many shortcomings of biomolecular microcarriers introduced by the presence of the solid surface of them. The suspension array, which used these macroporous hydrogel photonic beads as coding elements, showed obvious advantages in rapid biomolecular screening and highly sensitive detection. It is anticipated that this technology may also appeal to applications in drug screening, drug delivery, and medical diagnostics. Acknowledgment. We are grateful to the support of 333 Talent Project Foundation and Qing Lan Project of Jiangsu Province, Jiangsu Science and Technology Department (Grant No. BE2009148 and BE2008318), and National Science Foundation of China (Grant Nos. 50925309, 20703010). Y.-J.Z. thanks the China Scholarship Council. Supporting Information Available: Scheme and additional explanation of the macroporous hydrogel photonic beads fabrication, reflection peak changes of the hydrogel beads with different PEG concentrations, and reflection spectra and three-dimensional image of the hydrogel beads with six kinds of pore sizes. This material is available free of charge via the Internet at http://pubs.acs.org.

Langmuir 2010, 26(9), 6111–6114