Sensitive Discrimination and Detection of Prion ... - ACS Publications

Nov 1, 2010 - ... College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China, China Animal Health and Epidemiology Centers, Nat...
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Anal. Chem. 2010, 82, 9736–9742

Sensitive Discrimination and Detection of Prion Disease-Associated Isoform with a Dual-Aptamer Strategy by Developing a Sandwich Structure of Magnetic Microparticles and Quantum Dots Sai Jin Xiao,†,¶ Ping Ping Hu,‡ Xiao Dong Wu,⊥ Yan Li Zou,⊥ Li Qiang Chen,‡ Li Peng,‡ Jian Ling,†,# Shu Jun Zhen,† Lei Zhan,§ Yuan Fang Li,† and Cheng Zhi Huang*,†,§ Education Ministry Key Laboratory on Luminescence Real-Time Analysis, College of Chemistry and Chemical Engineering, College of Life Science, and College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China, China Animal Health and Epidemiology Centers, National Diagnostic Center for Exotic Animal Diseases, Qingdao 266032, China, Department of Applied Chemistry, East China Institute of Technology, Fuzhou 344000, China, and College of Chemical Science and Engineering, Yunnan University, Kunming 650091, China The major challenge of prion disease diagnosis at the presymptomatic stage is how to sensitively or selectively discriminate and detect the minute quantity of diseaseassociated prion protein isoform (PrPRes) in complex biological systems such as serum and brain homogenate. In this contribution, we developed a dual-aptamer strategy by taking the advantages of aptamers, the excellent separation ability of magnetic microparticles (MMPs), and the high fluorescence emission features of quantum dots (QDs). Two aptamers (Apt1 and Apt2), which can recognize their two corresponding distinct epitopes of prion proteins (PrP), were coupled to the surfaces of MMPs and QDs, respectively, to make MMPs-Apt1 and QDs-Apt2 ready at first, which then could be coassociated together through the specific recognitions of the two aptamers with their two corresponding distinct epitopes of PrP, forming a sandwich structure of MMPs-Apt1-PrP-Apt2-QDs and displaying the strong fluorescence of QDs. Owing to the different binding affinities of the two aptamers with PrPRes and cellular prion protein (PrPC), both of which have distinct denaturing detergent resistance, our dualaptamer strategy could be applied to discriminate PrPRes and PrPC successfully in serum. Further identifications showed that the present dual-aptamer assay could be successfully applied to the detection of PrP in 0.01% brain homogenate, about 1000-fold lower than that of commonly applied antibody-mediated assays, which can detect PrP just in 10% brain homogenate, indicating that the present designed dualaptamer assay is highly sensitive and adequate for * Corresponding author. Tel.: +86-23-68254659. Fax: +86-23-68367257. E-mail: [email protected]. † Education Ministry Key Laboratory on Luminescence Real-Time Analysis, College of Chemistry and Chemical Engineering, Southwest University. ‡ College of Life Science, Southwest University. § College of Pharmaceutical Sciences, Southwest University. ⊥ National Diagnostic Center for Exotic Animal Diseases. ¶ East China Institute of Technology. # Yunnan University.

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clinical diagnosis without isolation of target protein prior to assay. Prion disease, such as scrapie and bovine spongiform encephalopathy (BSE) in animals and Creutzfeldt-Jakob disease (CJD) and Kuru in humans,1 is a group of fatal neurodegenerative disorders and arises when cellular prion protein (PrPC) undergoes some conformation rearrangement, which leads the changes of physiochemical and biochemical properties.2 For example, PrPRes, the pathological isoform of PrPC, has much higher content of β-sheet than PrPC and has the tendency of aggregation, and then, PrPC is sensitive to proteinase K (PK) but PrPRes becomes mostly resistant to the digestion of PK.3 The emergence of vCJD in 1996 and its linkages to BSE has created enormous concerns since it might be potentially epidemic in humans,4 triggering urgent development of methods for prion disease diagnosis. The major challenge of prion disease diagnosis at the presymptomatic stage, however, is to discriminate and detect the minute quantities of PrPRes from the complex biological matrix such as blood and brain homogenate. Western blot assays and enzyme-linked immunosorbent assays (ELISAs) developed in the beginning are insufficiently sensitive, and up to now, there are only two strategies adopted to improve the sensitivity of disease diagnosis. The first one is based on the specific interaction of PrPRes with its specific antibodies with the aids of various spectroscopic techniques5-10 (1) Prusiner, S. B. Science 1997, 278, 245. (2) Campana, V.; Sarnataro, D.; Zurzolo, C. Trends Cell Biol. 2005, 15, 102– 111. (3) Linden, R.; Martins, V. R.; Prado, M. A. M.; Cammarota, M.; Izquierdo, I.; Brentani, R. R. Physiol Rev. 2008, 88, 673–728. (4) Grassi, J.; Maillet, S.; Simon, S.; Morel, N. Vet. Res. 2008, 39, 33. (5) Bellon, A.; Seyfert-Brandt, W.; Lang, W.; Baron, H.; ner, A. G.; Vey, M. J. Gen. Virol. 2003, 84, 1921–1925. (6) Englund, H.; Sehlin, D.; Johansson, A.-S.; Nilsson, L. N. G.; Gellerfors, P. r.; Paulie, S.; Lannfelt, L.; Pettersson, F. E. J. Neurochem. 2007, 103, 334– 345. (7) Coleman, B. M.; Nisbet, R. M.; Han, S.; Cappai, R.; Hatters, D. M.; Hill, A. F. Biochem. Biophys. Res. Commun. 2009, 380, 564–568. (8) Fujii, F.; Horiuchi, M.; Ueno, M.; Sakata, H.; Nagao, I.; Tamura, M.; Kinjo, M. Anal. Biochem. 2007, 370, 131–141. 10.1021/ac101865s  2010 American Chemical Society Published on Web 11/01/2010

such as fluorescence correlation spectroscopy (FCS), Fouriertransformed infrared spectroscopy (FT-IR), FIAsH fluorescence, and Raman spectroscopy. These spectroscopic methods, however, are cumbered for widespread use by the specificity and usefulness of Abs, expensive and sophisticated equipment, or the need of high level skill in measurements and interpretations of results. The second one is concentrated on how to amplify the minute quantities of PrPRes in samples through sodium phosphotungstic acid,11-13 plasminogen, or protein misfolding cyclic amplification (PMCA) technology and cell infective assay, which, however, is limited by the accuracy and feasibility and is time-consuming.14,15 In a step toward addressing the limitations mentioned above, herein, we develop an intelligent prion disease-associated isoform discrimination and detection assay by taking advantage of aptamers. Aptamers, single-stranded oligonucleotides with a length of tens of nucleotides, have found applications in molecular recognition,16 cancer diagnosis,17 protein detection,18 and cell imaging,19 owing to their intrinsic advantages such as excellent stability, reproducible synthesis, easy manipulation, and diagnostic potential.20 The majority of current aptamer-based protein assays that adopted single-aptamer binding configuration is insufficiently sensitive. In order to improve the sensitivity and specificity, dualaptamer binding configuration, in virtue of fluorescence resonance energy transfer (FRET) or proximity-dependent hybridization, has been applied for protein assays recently.21,22 In these assays, however, stringent experimental conditions and high level skill in experimental design are compulsory. For example, the number of DNA base pairs must be carefully and skillfully designed in the FRET assays in order to precisely control the distance between the donor and the acceptor.22–24 As the consequence, efficient fabrication of assays and the procedural complexity limit the applications of such assays in virtue of FRET or proximitydependent hybridization. PrP has two distinct binding epitopes for two different aptamer sequences. One aptamer, namely, Apt 1, can bind with the 23-90 epitope of PrP, and the other, namely, Apt2, the previously designated 4C6 by Bibby et al., can bind with the 90-231 epitope (9) Krafft, C.; Steiner, G.; Beleites, C.; Salzer, R. J. Biophotonics 2009, 2, 13– 28. (10) Soto, C. Nat. Rev. 2004, 2, 809–819. (11) Fischer, M. B.; Roeckl, C.; Parizek, P.; Schwarz, H. P.; Aguzzi, A. Nature 2000, 408, 479–483. (12) Maissen, M.; Roeckl, C.; Glatzel, M.; Goldmann, W.; Aguzzi, A. Lancet 2001, 357, 2026–2028. (13) Wadsworth, J. D. F.; Joiner, S.; Hill, A. F.; Campbell, T. A.; Desbruslais, M.; Luthert, P. J.; Collinge, J. Lancet 2001, 358, 171–180. (14) Saborio, G. P.; Permanne, B.; Soto, C. Nature 2001, 411, 810–813. (15) Vorberg, I.; Raines, A.; Story, B.; Priola, S. A. J. Infect. Dis. 2004, 189, 431–439. (16) Chen, H. W.; Medley, C. D.; Sefah, K.; Shangguan, D.; Tang, Z. W.; Meng, L.; Smith, J. E.; Tan, W. H. ChemMedChem 2008, 3, 991–1001. (17) Huang, Y.-F.; Chang, H.-T.; Tan, W. H. Anal. Chem. 2008, 80, 567–572. (18) Davis, J. J.; Tkac, J.; Humphreys, R.; Buxton, A. T.; Lee, T. A.; Ko Ferrigno, P. Anal. Chem. 2009, 81, 3314–3320. (19) Bagalkot, V.; Jon, S.; Kantoff, P. W.; Langer, R.; Farokhzad, O. C. Nano Lett. 2007, 7, 3065–3070. (20) Phillips, J. A.; Lopez-Colon, D.; Zhu, Z.; Xu, Y.; Tan, W. Anal. Chim. Acta 2008, 621, 101–108. (21) Lee, W.; Obubuafo, A.; Lee, Y.-I.; Davis, L. M.; Soper, S. A. J. Fluoresc. 2010, 20, 203–213. (22) Zhang, Y.-L.; Huang, Y.; Jiang, J.-H.; Shen, G.-L.; Yu, R.-Q. J. Am. Chem. Soc. 2007, 129, 15448–15449. (23) Heyduk, E.; Heyduk, T. Anal. Chem. 2005, 77, 1147–1156. (24) Rinker, S.; Ke, Y.; Liu, Y.; Chhabra, R.; Yan, H. Nature Nanotechnol. 2008, 3, 418–422.

of PrP. The binding ability of the two aptamers, however, depends on the guanidinium denaturation induced conformation of PrPC and PrPRes.25 By making use of the different binding affinities of aptamers, especially Apt2, to denatured PrPC and PrPRes, herein, we developed a dual-aptamer assay for the discrimination and detection of PrPRes. Different from other reports, which need tremendous and complicated test work for accurate distance-control to ensure high efficiency of resonance energy transfer in FRET assays, our present contribution makes use of the exceptional separation ability of magnetic microparticles (MMPs), the high emission quality of quantum dots (QDs), and the specific recognitions of dual-aptamers with their two distinct binding epitopes of PrP target, avoiding FRET and proximity-dependent hybridization so as to make it possible for the sensitive prion disease-associated isoform discrimination and detection in serum and brain homogenate samples. With this strategy, prion disease-associated protein isoform discrimination and detection could be achieved first by addressing two aptamers’ recognition for distinct epitopes of the target and the avoidance of distance-control for the FRET process. EXPERIMENTAL SECTION Apparatus. Fluorescence and absorption measurements were made with a Hitachi F-2500 fluorescence spectrophotometer and a U-3010 spectrophotometer (both were from Hitachi, Tokyo, Japan), respectively. The circular dichroism (CD) spectra were obtained by a J-810 spectropolarimeter (JASCO Co., Japan), and a KQ-100 ultrasonic processor (Kunshan Ultrasonic Instruments Factory, Jiangsu, China) was employed for the dissociation of MMPs. The fluorescence imaging was acquired with a IX81 microscope with a 10× objective (Olympus, Japan). Reagents. Streptavidin modified quantum dots (QDs) were commercially available from Jiayuan Quantum Dot Co. Ltd. (Wuhan, China). Two aptamers, Apt1, NH2-CTT ACG GTG GGG CAA TT, and Apt2, Bio-GTT TTG TTA CAG TTC GTT TCT TTT CCC TGT CTT GTT TTG TTG TCT, were selected by Takemura and Bibby, respectively,25,26 and synthesized by Sangon Tech. Ltd. (Shanghai, China) without further purification. Gdn-HCl was purchased from Genview (USA). Ultrapure water (18.2 MΩ, LD-50G-E Lidi Ultra Pure Waters System, Chongqing, China) was used throughout. Pepsin and bovine serum albumin (BSA) were purchased from Shanghai Biochemicals (Shanghai, PRC). Other commercial reagents such as sodium chloride and nickel chloride were analytical reagent grade without further purification. Coupling of Magnetic Microparticles with Apt1. Magnetic microparticles (MMPs) of Fe3O4 were prepared according to references (for details, please see Supporting Information). For coupling with Apt1, the suspension of MMPs (10 mg) in 0.01 mol/L PBS (pH 7.4) with 2.5% (v/v) glutaraldehyde (GA) was incubated at 37 °C under gentle stirring for 1 h, washed three times with 0.01 mol/L PBS to remove redundant GA, and then equilibrated overnight with 1.4 nmol (500 µL) NH2-Apt1 by shaking at 200 rpm at 37 °C. The MMPs-Apt1 complexes were (25) Bibby, D. F.; Gill, A. C.; Kirby, L.; Farquhar, C. F.; Bruce, M. E.; Garson, J. A. J. Virol. Methods 2008, 151, 107–115. (26) Takemura, K.; Wang, P.; Vorberg, I.; Surewicz, W.; Priola, S. A.; Kanthasamy, A.; Pottathil, R.; Chen, S. G.; Sreevatsan, S. Exp. Biol. Med. 2006, 231, 204–214.

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washed several times and finally resuspended in 10 mL of 0.01 mol/L PBS (pH 7.4). The coupling efficiency was determined by the decreased absorbance at 260 nm of Apt1 in aqueous medium (refer to Supporting Information, S1). Purification of Recombinant Human Cellular Prion Protein (rPrP) and Conversion of rPrP to PrPRes. The rPrP was purified according to the method developed by Xiao’s group,27 and the plasmid of recombinant human prion protein (23-231) was constructed and expressed in Escherichia coli BL21 (DE3). For protein purification, a fresh overnight culture was induced by 50 µg/mL isopropyl-d-thiogalactopyranoside (Sigma, USA). After 6 h, the cells were harvested by centrifugation and sonicated in lysis buffer (50 mmol/L NaH2PO4, 300 mmol/L NaCl, and 10 mmol/L imidazole, pH 8.0). The resulting solution was then denatured in 6 mol/L guanidine hydrochloride (Gdn-HCl) overnight and purified by nickel-nitrilotriacetic acid agarose resin (Invitrogen, Germany). The purified prion protein was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and CD spectra, and the concentration of purified prion proteins was determined with the Bradford Protein Assay Kit (TianGen, Beijing). The conversion of rPrP to PrPRes was achieved according to Bocharova et al.28 Namely, a stock solution of 73.8 µmol/L R-form rPrP was diluted to 22 µmol/L and incubated at 37 °C with 1 mol/L Gdn-HCl, 3 mol/L urea, and 150 mmol/L NaCl at pH 4.0 in 20 mmol/L sodium acetate buffer for 48 h and then dialyzed with 20 mmol/L sodium acetate buffer. The conformation of PrPRes was confirmed by CD spectra (see in Supporting Information, S2). Discrimination and Detection of PrP. Two sample solutions, both were, respectively, in native state (NS) and in denatured state (DS) pretreated with 4 mol/L Gdn-HCl at 83 °C for 10 min and were prepared prior to use. Sixty microliters of MMPs-Apt1, 30 µL of 1.0 mg/mL BSA, and certain concentrations of samples were incubated in 0.02 mol/L phosphate buffer (PB, pH 6.1) and 0.2 mol/L NaCl for 30 min at room temperature. The solution was washed with PB three times to remove abundant proteins by magnetic separation, and then 40 µL of QDs-Apt2 were added into the resuspension and reacted in the mixture of 0.02 mol/L PB (pH 7.4) and 0.2 mol/L NaCl at room temperature with end-overend rotation for 60 min. After completion of the reaction, an external magnet was used to eliminate free QDs-Apt2. The fluorescence of the formed MMPs-Apt1-PrP-Apt2-QDs sandwich structure was measured with the excitation at 365 nm and emission at 608 nm, which is predominant from the QD emission. For the detection of PrPRes, the sample was pretreated with 4.0 mol/L of Gdn-HCl at 83 °C for 10 min to eliminate the influence of PrPC. Detection of Brain Homogenate with Present DualAptamer and Western-Blot Assays. The preparations of mice brain homogenate were detailed in the Supporting Information. For PrPC analysis with our designed dual-aptamer assay, two brain homogenate samples in both native state (NS) and denatured state (DS) were prepared, and the later one was pretreated with 4 mol/L of Gdn-HCl at 83 °C for 10 min prior (27) Yu, S.-L.; Lei, J.; SY, M.-S.; Mei, F.-H.; Kang, S.-L.; Sun, G.-H.; Tien, P.; Wang, F.-S.; Xiao, G.-F. Eur. J. Hum. Genet. 2004, 12, 867–870. (28) Bocharova, O. V.; Breydo, L.; Parfenov, A. S.; Salnikov, V. V.; Baskakov, I. V. J. Mol. Biol. 2005, 346, 645–659.

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Figure 1. Schematic presentation of our dual-aptamer strategy for sensitive discrimination of prion disease-associated isoform. Two aptamers (Apt1 and Apt2) that can recognize their corresponding epitopes of PrP were first coupled to magnetic microparticles (MMPs) and quantum dots (QDs), respectively, making MMPs-Apt1 and QDsApt2 available. In the presence of the PrP target proteins, both MMPsApt1 and QDs-Apt2 can bind to PrP, forming a fluorescent sandwich structure of MMPs-Apt1-PrP-Apt2-QDs. As PrPRes is denaturingdetergent resistant such as guanidine hydrochloride (Gdn-HCl) while PrPC is sensitive to the denaturing detergent, the fluorescence of MMPs-Apt1-PrPRes-Apt2-QDs gets enhanced if PrPRes denatured since the epitope of Apt2 becomes more accessible (shown as an open pocket of the third one in the right column), while that of MMPsApt1-PrPC-Apt2-QDs disappeared if PrPC denatured since the two epitopes have been destroyed (shown as the closed pocket and the random coiled state of the fourth one in the right column). The numbers 23, 90, and 231 in PrP represents the 23rd, 90th, and 231st amino acid, respectively, and Apt 1 can bind with the 23-90 epitope of PrP, while Apt2 can bind with the 90-231 epitope of PrP.

to use. For Western Blot analysis, native and denatured brain homogenate samples were also prepared, respectively, but the later one was pretreated with proteinase K (Amresco, USA) at 37 °C for 1 h prior to use. The native and denatured 10%, 1%, and 0.5% brain homogenates were added to 2× loading buffer (100 mM Tris-HCl, 10% glycerol, 4% SDS, 200 mM dithiothreitol (DTT), pH 6.8), boiled for 5 min, separated on 12% Precise Protein Gels (Pierce, Thermo Scientific, USA), and ran until the blue marker went to the end of the gel. Then, the samples were transferred to the PVDF film (Millipore, USA) with a semidry method and blocked with TBST buffer overnight (10 mM Tris, 150 mM sodium chloride, 0.1% Tween 20, 2% BSA, pH 7.4). After being incubated with anti-PrP monoclonal antibody at room temperature for 1 h and washed with TBST 5 times, the samples were then incubated with peroxidase conjugated anti-mouse IgG at room temperature for 1 h and washed with TBST 5 times. The samples were finally detected and pictured with FluorChem FC2 (Alpha innoteck, USA) after

Figure 2. Fluorescence spectrum of dual-aptamer strategy with PrPC (red curve, 1.35 × 10-6 M) and PrPRes (blue curve, 1.37 × 10-6 M), respectively. The black curve represents the blank control of dualaptamer strategy.

Figure 3. (A) Plots of fluorescence emission changes of dual-aptamer assay without (dot line) and with 10A spacer (solid line) as a function of PrPRes. (B) BSA as a blocking agent for 8.46 × 10-7 mol/L PrPRes (dotted line) compared to the non-PrP control.

being incubated with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Thermo Scientific, USA). Reuse of MMPs-Apt1 Probe. The MMPs-Apt1-PrP-Apt2-QDs sandwich structure was incubated in 1 mol/L NaOH at 50 °C for 10 min and washed three times with 0.01 mol/L PB buffer (pH 6.1) by an external magnet; the regenerated MMPs-Apt1 probe could be reused or stored at 4 °C. RESULTS AND DISCUSSION Dual-Aptamer Assay Design. Our strategy is showed in Figure 1, which starts off with the chemical coating of both MMPs and QDs with aptamer 1 (Apt1) and aptamer 2 (Apt2), respectively. Then, the prepared MMPs-Apt and QDs-Apt2 probes can form a sandwich structure of MMPs-Apt1-PrP-Apt2-QDs in the presence of the PrP target since both MMPs-Apt1 and QDs-Apt2 can simultaneously bind to their corresponding epitopes of PrP. It is known that MMPs have excellent separation ability and QDs have high photoluminescence quality; then, the sandwich structure is highly fluorescent in the aqueous mediums, and it can be separated in the external magnetic fields. The discrimination of PrPC and PrPRes is based on the different binding affinities of the two aptamers to the DS-PrP. PrPC is sensitive to denaturing detergent such as guanidine hydrochloride (Gdn-HCl), and PrPRes is denaturing-detergent resistant,; therefore, the fluorescence of MMPs-Apt1-PrPResApt2-QDs gets enhanced if PrPRes denatured since the epitope of Apt2 become more accessible (shown as an wide open pocket of the third one in the right column in Figure 1), while that of MMPs-Apt1-PrPC-Apt2-QDs disappeared if PrPC denatured since the two epitopes for both Apt1 and Apt 2 have been destroyed (shown as the closed pocket and the random coiled state of the fourth one in the right column in Figure 1). To achieve the prion disease-associated isoform discrimination and detection by the designed dual-aptamer assay, the primary compulsory step is to identify whether the two aptamers could coassociate with PrP simultaneously or not through the specific recognition with their distinct epitopes of PrP. As shown in Figure 2, increased fluorescence emission of QDs was observed when MMPs-Apt1 and QDs-Apt2 were incubated with certain amounts of NS-PrPC or NS-PrPRes, illuminating that the two aptamers are able to coassociate with PrP to form a sandwich structure of MMPs-Apt1-PrP-Apt2-QDs and our design is really reasonable. Spacer and Blocking Agent. It is well-known that the sensitivities of DNA and protein assays could be improved by the

incorporation of spacers such as olig(dT) and carbon spacers.29 Considering that Apt2 has 45-mer of base, which is long enough for the coupling, thus herein, we only consider the incorporation of single-stranded DNA oligomers as spacers into Apt1. It is obvious that Apt1 with 10A spacer shows a stronger fluorescence response than that of the unmodified Apt1, and the sensitivity of the assay with the incorporation of spacer is about 1000-fold higher than that without spacer (Figure 3A and Figure S3, Supporting Information). In addition, the results imply that the incorporation of spacer into Apt1 does not disturb the interaction between aptamers and targets through intramolecular T-A base pairing, and also, the 10A spacer incorporated into Apt1 endows the aptamer on the MMP surface to be more accessible to PrP. In order to avoid nonspecific absorption of MMPs and promote the binding efficiency of aptamers with PrP, blocking agents such as BSA and PEG should be controlled in aqueous medium.30 Experiments showed that the fluorescence emission increased dramatically with BSA acting as blocking agent (Figure 3B), which might be due to the nonspecific absorption of the blocking agent that can efficiently prevent target proteins and QDs-Apt2 to the surface of MMPs, whose surface has an unmodified aldehyde group and amido.31 However, excess amount of blocking agent decreases the binding affinity of aptamers owing to the steric hindrance caused by excessive blocking agent. Discrimination and Detection of PrPRes. Both NS-PrPC and NS-PrPRes have two epitopes for the specific binding of the two aptamers, Apt1 and Apt2, and thus, the fluorescence signals could be observed easily (Figure 4A). However, the affinities of both DS-PrPC and DS-PrPRes, which were prepared by introducing guanidine hydrochloride (Gdn-HCl) since PrPC and PrPRes display different denaturation curves,32-34 are different with the two aptamers since the epitopes of both DS-PrPC and DS-PrPRes are distinct, and thus, distinct fluorescence signal responses could be obtained (Figure 4A). For PrPRes, owing to its GdnHCl resistance, the binding affinity of Apt2 to DS-PrPRes is higher than that to NS-PrPRes as the epitope of the DS-PrPRes for Apt2 becomes more accessible (as indicated of wide open (29) Lao, Y.-H.; Peck, K.; Chen, L.-C. Anal. Chem. 2009, 81, 1747–1754. (30) Hu, P.; Huang, C. Z.; Li, Y. F.; Ling, J.; Liu, Y. L.; Fei, L. R.; Xie, J. P. Anal. Chem. 2008, 80, 1819–1823. (31) Bruce, I. J.; Sen, T. Langmuir 2005, 21, 7029–7035. (32) Chang, B.; Miller, M. W.; Bulgin, M. S.; Sorenson-Melson, S.; Balachandran, A.; Chiu, A.; Rubenstein, R. J. Neuroimmunol. 2008, 205, 94–100. (33) Leliveld, S. R.; Korth, C. J. Neurosci. Res. 2007, 85, 2285–2297. (34) Novitskaya, V.; Makarava, N.; Bellon, A.; Bocharova, O. V.; Bronstein, I. B.; Williamson, R. A.; Baskakov, I. V. J. Biol. Chem. 2006, 281, 15536–15545.

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pocket of the third one in the right column in Figure 1), giving a stronger fluorescence signal than that of NS-PrPRes. Conversely, the epitopes of DS-PrPC for Apt1 and Apt2 were destroyed owing to the pretreatment with Gdn-HCl (showing as the close pocket and the mess structure of the fourth one in the right column of Figure 1), and therefore, no fluorescence could be observed in an aqueous medium. These results are identical to that of Bibby et al.,25 showing that the guanidinium denaturation reduces the aptamer binding ability to PrPC and increases the ability to PrPRes. The strong fluorescence emission could also be imaged by a fluorescence microscope; as shown in Figure 4B, both NS-PrPC and NS-PrPRes display strong fluorescence, and DS-PrPRes displays enhanced fluorescence compared with that of NSPrPRes. This resistant result of PrPRes to Gdn-HCl is identical to the other observations in conformational discrimination.34–36 In order to accomplish prion disease diagnosis at a presymptomatic stage, dynamic response, sensitivity, and specificity of the present assay have been validated. As shown in Figure 5,

sequential increases of fluorescence emission were observed when MMPs-Apt1 and QDs-Apt2 were incubated together with increasing content of PrPRes. In the range 0.8-306.0 nmol/L, there is a good linear relationship between the enhanced fluorescence and the concentration of PrPRes with the correlation coefficient of 0.998 (n ) 11). Discrimination and Detection of PrPRes in Serum. For prion diseases diagnosis, it is the top priority to develop a method for blood tests.37 In order to validate the feasibility of the dualaptamer assay for PrPRes discrimination in a complex biological system, which needs a far more demanding test bed for protein sensing than that in buffer solution,38 serum was employed as the medium for the reason that fibrin has no cross-reactivity for the discrimination of PrPRes (see Supporting Information, Figure S4). For the discrimination of PrPC and PrPRes in FBS, the concentration of PrPRes was kept as 5.5 × 10-7 mol/L (∼ 3.97 µg/mL) while FBS was kept as 3.97 mg/mL. In Figure 6A, the abscissa represents the molar ratio of PrPC and PrPRes (PrPC/PrPRes). It can be seen that the fluorescence of a native mixture of PrPC and fetal bovine serum (FBS) gets increased dramatically, while that of the denatured mixture of PrPC and FBS disappeared. On the contrary, the fluorescence of either the native or the denatured mixture of PrPC, PrPRes, and FBS gets increased. Furthermore, the fluorescence of denatured mixture of PrPC, PrPRes, and FBS gets enhanced compared with that of the native mixture. All the results identify that our present dual-aptamer strategy could be applied for PrPRes discrimination in serum. The PrPRes detection in serum was demonstrated (Figure 6B), showing that the dual-aptamer assay can readily achieve PrPRes detection even if a 1000-fold or higher excess of FBS is present. Noticeably, compared to the 1:0 ratios of PrPRes/FBS (w/w), the fluorescence emission gets increased with the addition of FBS by keeping the ratios of PrPRes/FBS at 1:1-1: 10, but the fluorescence emission gets decreased gradually if the ratio of PrPRes/FBS is higher than 1:10. We suppose that the fluorescence increase is due to the nonspecific interaction between FBS and QDs (see in Supporting Information, Figure S5) since there are surface defects and dangling bonds of quantum dots.39 If the ratio of PrPRes/FBS is higher than 1:10, however, lots of FBS result in a strong effect on the interaction between PrPRes and the two aptamers. The high selectivity shown in Figure 6B is adequate for clinical diagnosis and serum proteomic analysis, in which it is not necessary to separate or purify the target protein prior to assays. Detection of PrPC in Mice Brain Homogenate. To further demonstrate the feasibility of present dual-aptamer assay in animal derived sample, mice brain homogenate was analyzed. As shown in Figure 7A, the fluorescence emission gets increased if MMPsApt1 and QDs-Apt2 were incubated with brain homogenate, and the PrPC detection in 0.01% brain homogenate could be successfully achieved. The fluorescence disappeared if brain homogenate was pretreated with Gdn-HCl, which is the same as the brain homogenate pretreated with proteinase K (PK;

(35) Peretz, D.; Williamson, R. A.; Matsunaga, Y.; Serban, H.; Pinilla, C.; Bastidas, R. B.; Rozenshteyn, R.; James, T. L.; Houghten, R. A.; Cohen, F. E.; Prusiner, S. B.; Burton, D. R. A. J. Mol. Biol. 1997, 273, 614–622. (36) Sun, Y.; Breydo, L.; Makarava, N.; Yang, Q.; Bocharova, O. V.; Baskakov, I. V. J. Biol. Chem. 2007, 282, 9090–9097.

(37) Brown, P. Vox Sang. 2005, 89, 63–70. (38) De, M.; Rana, S.; Akpinar, H.; Miranda, O. R.; Arvizo, R. R.; Bunz, U. H. F.; Rotello, V. M. Nature Chem. 2009, 1, 461–465. (39) Wang, Q.; Kuo, Y. C.; Wang, Y. W.; Shin, G.; Ruengruglikit, C.; Huang, Q. R. J. Phys. Chem. B 2006, 110, 16860–16866.

Figure 4. (A) Fluorescence response of the sandwich structure of MMPs-Apt1-PrP-Apt2-QDs in native state (NS) and denatured state (DS). Black curve represents the control; red and green ones represent that in the presence of 1.52 × 10-6 mol/L PrPC and DSPrPC, while blue and cyan ones represent that in the presence of 1.14 × 10-6 mol/L PrPRes and DS-PrPRes. The inset is the fluorescence changes, and the light gray columns represent NS while the dark gray ones are DS. (B) Fluorescence imaging of the two isoforms of prion diseases in native (upper row) and denatured state (the bottom row) of PrPC (left column) and PrPRes (right column). PrPC and PrPRes were kept as 1.52 × 10-6 and 1.14 × 10-6 mol/L, respectively. QDs 605 were excited with a JC12 V100W Halogen bulb, and the emission of fluorescence was measured with Barrier Filter BA595-615 nm.

Figure 5. Detection of DS-PrPRes with the dual-aptamer strategy. The linear equation is ∆IF ) 2.37 + 0.30 cPrPRes in the range of 0.8-306.0 nmol/L with the correlation coefficient (r) of 0.998 (n ) 11).

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Figure 6. Discrimination and detection of PrPRes. (A) Discrimination of PrPRes in serum by keeping PrPRes as 5.5 × 10-7 mol/L (∼3.97 µg/mL). The abscissa represents the ratio of PrPC to PrPRes. The denatured state (DS) sample was obtained by pretreating PrP with 4 mol/L Gdn-HCl. (B) Detection of PrPRes in serum. PrPRes was kept as 5.96 µg/mL (∼0.84 × 10-6 mol/L), and FBS was diluted with 0.01 mol/L PB (pH 7.4). The light gray columns represent the fluorescence intensities of dual-aptamer assay control while the dark gray ones present that of PrPRes in FBS. All the samples were pretreated with 4 mol/L Gdn-HCl at 83 °C for 10 min in 20 mmol/L acetate sodium buffer (pH 4.0) to mimic the condition of PrPRes discrimination and detection.

Figure 7. Results of brain homogenate analysis. (A) Results with present dual-aptamer assay. The light gray columns represent brain homogenate in a native state while the heavy gray ones represent brain homogenate that is pretreated with 4 mol/L Gdn-HCl at 83 °C for 10 min. (B) Results of Western blot analysis. M represents protein marker; PK+ represents the brain homogenates pretreated with proteinase K at room temperature for 1 h, and PK represents the brain homogenate untreated. The picture was captured by FluorChem FC2 (Alpha innoteck).

see in Supporting Information, Figure S6). These results suggest that our present dual-aptamer assay could be applied to the direct detection of PrPC in animal derived samples and might further be used for the detection of PrPRes from an animal derived sample since the influence of PrPC could be successfully eliminated by Gdn-HCl denaturation. With the purpose of illustrating the high sensitivity of our present dual-aptamer assay, mice brain homogenate was also analyzed with Western blot analysis. As shown in Figure 7B, PrPC could be detected just only in 10% brain homogenate and could not be detected in 1%, 0.5%, and PK pretreated samples, indicating that the sensitivity of our dual-aptamer assay gets highly improved and is about 1000-fold higher than the Western blot assay. Reuse of MMPs-Apt1 Probe. Since only involved in a surface-tethered monolayer of Apt1 without preferred conformation, the MMPs-Apt1 probe could be readily regenerated. As shown in Figure 8, the sensitivity of MMPs-Apt1 could be preserved even after five cycles. The dissociation of PrP from MMPs-Apt1 could be confirmed by the increasing fluorescence emission of the aqueous medium after being separated by an external magnet (refer to Supporting Information, Figure S7), and the fluorescence measurements showed that reused MMPs-Apt1 probe has small peaks at around 608 nm, which might be ascribed to two possibilities. One is that there is the incomplete dissociation

Figure 8. Reuse of MMPs-Apt1. Good signal response could be achieved over five cycles by keeping PrPRes at 6.84 × 10-6 mol/L.

of PrP from the MMPs-Apt1 surface, which can be avoided by further addition of NaOH. The other is that there is the nonspecific absorption of QDs-Apt2 on the surface of MMPs-Apt1 since the denaturation with NaOH is involved not only in PrP but also in the blocking agent of BSA. CONCLUSION Two distinct PrP-binding aptamers (Apt1 and Apt2) are introduced in our assay to tackle the sensitivity and specificity issue in prion disease diagnosis. It is obvious that the two aptamers for the distinct epitopes of prion protein can be applied in Analytical Chemistry, Vol. 82, No. 23, December 1, 2010

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conformational discrimination and prion disease-associated isoform detection. On the basis of the different binding affinities of aptamers and the distinct resistances of PrPC and PrPRes to GdnHCl, our dual-aptamer assay can achieve PrPRes discrimination successfully in buffer solution as well as in serum with high ratio of signal-to-noise. The sensitivity of the dual-aptamer assay can be improved to 85.5 pmol/L when a olig(dA) spacer was incorporated into Apt1. Moreover, the designed dual-aptamer assay can be applied for diseases-associated isoform detection even if a 1000-fold or higher excess of brain homogenate is present, indicating that the dual-aptamer assay is adequate for diagnosis without isolation and purification of target proteins prior to assays. The high sensitivity and selectivity of the dualaptamer assay can be improved by the avidity effect resulting from two coassociated aptamers that attain a synergistic binding event. As the specificity issues have been solved successfully by the employment of dual-aptamer recognition in the assay, further work could be concentrated mainly on the improvement of the sensitivity to make our dual-aptamer assay suitable for direct clinical diagnosis, which might be assisted with the

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amplification of the existing signal and concentrated with the minute quantities of PrPRes in the sample by MMPs-Apt1. ACKNOWLEDGMENT We thank Dr. Geng Fu Xiao from Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China, for providing the plasmid encoding recombinant human (rhPrPC) (23-231) as a gift. The authors show their great appreciation for the financial support of the Ministry of Science and Technology of the People’s Republic of China (No. 2006CB933100 and 2011CB933600) and the National Natural Science Foundation of China (NSFC, 20775061). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review July 13, 2010. Accepted October 17, 2010. AC101865S