DNA Encapsulating Liposome Based Rolling Circle Amplification

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DNA Encapsulating Liposome Based Rolling Circle Amplification Immunoassay as a Versatile Platform for Ultrasensitive Detection of Protein Li-Juan Ou, Si-Jia Liu, Xia Chu,* Guo-Li Shen, and Ru-Qin Yu State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China A novel rolling circle amplification (RCA) immunoassay based on DNA-encapsulating liposomes, liposome-RCA immunoassay, was developed for ultrasensitive protein detection. This technique utilized antibody-modified liposomes with DNA prime probes encapsulated as the detection reagent in the sandwiched immunoassays. The DNA prime probes were released from liposomes and then initiated a linear RCA reaction, generating a long tandem repeated sequences that could be selectively and sensitively detected by a microbead-based fluorescence assay. The developed technique offered very high sensitivity due to primary amplification via releasing numerous DNA primers from a liposome followed by a secondary RCA amplification. A biobarcode design was incorporated in the technique, which allowed the strategy to be directly implemented for multiplex assay of multiple proteins. Also, the technique allowed easy preparation of the DNAcarrying antibody reagent and the implementation with simple instrumentation. The technique was demonstrated for the determination of prostate-specific antigen (PSA), a highly selective biomarker associated with prostate cancer. The results revealed that the technique exhibited a dynamic response to PSA over a 6-decade concentration range from 0.1 fg mL-1 to 0.1 ng mL-1 with a limit of detectionaslowas0.08fgmL-1 andahighdose-response sensitivity. The liposome-RCA immunoassay holds great promise as a versatile, sensitive, and robust platform to combine the nucleic acid amplification with immunoassay for ultrasensitive protein detection. Sensitive detection of proteins has become a critical need for proteomics and clinical diagnostics.1 Typically, protein assay is performed using the antibody-based immunoassay system, in which the protein targets are specifically bound by their antibodies and then the binding event is detected via some signal-reporting labels. In this system, the assay performance is primarily dependent on detection sensitivity and nonspecific adsorption of the signal-reporting labels. Therefore, the development of signalreporting labels with maximized signal amplification and minimized nonspecific adsorption is crucial for performance improvement in the immunoassay system. In general, high-sensitivity * To whom correspondence should be addressed. E-mail: [email protected]. Phone: +86-731-88821916. Fax: +86-731-88821916 . (1) Gosling, J. P. Clin. Chem. 1990, 36, 1408–1427.

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immunoassay labels involve the use of certain signal amplification strategies by which a single signal-reporting tag is able to incorporate numerous detectable elements. Typical high-sensitivity immunoassay tags include enzymes2,3 or their polymeric forms4 as well as nanoparticles such as gold colloid,5,6 dye-doped nanoparticles,7,8 and quantum dot.9 Notwithstanding the importance of these techniques, the signal amplification capacity of these immunoassay labels is still limited. An attractive alternative is the use of oligonucleotide sequences as the immunoassay labels, considering the enormous capabilities of oligonucleotides in sitespecific labeling,10 as well as sequence-specific biobarcoding,11 amplification,12 and isolation.13 Along the direction of implementation of oligonucleotidebased immunoassay labels, immunopolymerase chain reaction (immuno-PCR)14,15 and immunorolling circle amplification (immuno-RCA),16-18 are of dominant significance. These techniques are able to offer very high detection sensitivity. However, both immuno-PCR and immuno-RCA require the conjugates of antibody and DNA to convert the antigen-antibody binding events into nucleic acid based assay. These conjugates are routinely prepared using the biotin-streptavidin bridge or direct covalent linkage of DNA to antibody. Such procedures (2) Kucera, E.; Kainz, C.; Tempfer, C.; Zeillinger, R.; Koelbl, H.; Sliutz, G. Anticancer Res. 1997, 17, 4735–4737. (3) Butler, J. E. J. Immunoassay 2000, 21, 165–209. (4) Wang, J.; Liu, G.; Jan, M. R. J. Am. Chem. Soc. 2004, 126, 3010–3011. (5) Nam, J. M.; Thaxton, C. S.; Mirkin, C. A. Science 2003, 301, 1884–1886. (6) Nam, J. M.; Stoeva, S. I.; Mirkin, C. A. J. Am. Chem. Soc. 2004, 126, 5932– 5933. (7) Smith, J. E.; Medley, C. D.; Tang, Z.; Shangguan, D.; Lofton, C.; Tan, W. Anal. Chem. 2007, 79, 3075–3082. (8) Wang, L.; Tan, W. Nano. Lett. 2006, 6 (1), 84–88. (9) Hansen, J. A.; Wang, J.; Kawde, A.-N.; Xiang, Y.; Gothelf, K. V.; Collins, G. J. Am. Chem. Soc. 2006, 128, 2228–2229. (10) Zhang, Y. L.; Huang, Y.; Jiang, J. H.; Shen, G. L.; Yu, R. Q. J. Am. Chem. Soc. 2007, 129, 15448–15449. (11) Hill, H. D.; Mirkin, C. A. Nat. Protoc. 2006, 1, 324–336. (12) Schweitzer, B.; Kingsmore, S. Curr. Opin. Biotechnol. 2001, 12, 21–27. (13) Miao, J. M.; Cao, Z. J.; Zhou, Y.; Lau, C. W.; Lu, J. Z. Anal. Chem. 2008, 80, 1606–1613. (14) Sano, T.; Smith, C. L.; Cantor, C. R. Science 1992, 258, 120–122. (15) Ruzicka, V.; Marz, W.; Russ, A.; Gross, W. Science 1993, 260, 698–699. (16) Schweitzer, B.; Wiltshire, S.; Lambert, J.; O’Malley, S.; Kukanskis, K.; Zhu, Z.; Kingsmore, S. F.; Lizardi, P. M.; Ward, D. C. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 10113–10119. (17) Schweitzer, B.; Roberts, S.; Grimwade, B.; Shao, W.; Wang, M.; Fu, Q.; Shu, Q.; Laroche, I.; Zhou, Z.; Tchernev, V. T.; Christiansen, J.; Velleca, M.; Kingsmore, S. F. Nat. Biotechnol. 2002, 20, 359–365. (18) Raghunathan, A.; Sorette, M. P.; Ferguson, H. R.; Piccoli, S. P. Clin. Chem. 2002, 48 (10), 1853–1855. 10.1021/ac901786m CCC: $40.75  2009 American Chemical Society Published on Web 10/30/2009

are frequently time-consuming and labor-intensive because they involve multiple incubation and separation steps. Moreover, the negatively charged DNA labels are susceptible to nonspecific adsorption on positively charged sites of capture antibodies and blocking agents, which increases the risk of false positive results. Additionally, the antibody-DNA conjugates are less stable during the assay, since impurities present in the sample that are incompletely removed by the wash steps can chemically or enzymatically degrade the DNA labels. This introduces an extra risk of false negative results. Therefore, the search of novel strategies for ultrasensitive DNA-based immunoassays still remains a challenge. Liposomes, spherical vesicles composed of a phospholipid bilayer surrounding an aqueous cavity, represent a promising module for ultrasensitive DNA-based immunoassays. Because of their ability to carry varying agents in the aqueous cavity for signal reporting and to tether different functional groups on the surface for biomolecular conjugation, this module have been adapted to a variety of immunoassay protocols. Prominent examples include lateral flow or flow injection immunoassays based on liposomes entrapping dyes or bioluminescent proteins,19-30 and immunosensors based on liposomes containing electroactive or electrogenerated chemiluminescent species.31-33 These liposome-based immunoassays offer the possibility of primary signal amplification via the bearing of numerous reporter molecules in a single liposome. To obtain further sensitivity enhancement, a secondary signal amplification strategy has been proposed by using DNAencapsulating liposomes. The released DNA sequences with hapten tags are first captured by the cDNA probe and then detected using the anti-hapten antibody-modified liposomes with dyes encapsulated.34 Also, an ultrasensitive liposome-PCR immunoassay has been developed by use of liposomes encapsulating double-stranded DNA. Then, the released double-stranded DNA is quantified by real-time PCR.35 Although the requirement for thermal cycling and experimental expertise in quantitative PCR may restrict the wide applications of liposome-PCR immunoassay, this strategy reveals the capacity of DNA-encapsulating liposomes (19) Ho, J.-A. A.; Wauchope, R. D. Anal. Chem. 2002, 74, 1493–1496. (20) Ahn-Yoon, S.; DeCory, T. R.; Baeumner, A. J.; Durst, R. A. Anal. Chem. 2003, 75, 2256–2261. (21) Zaytseva, N. V.; Richard, A.; Montagna, R. A.; Baeumner, A. J. Anal. Chem. 2005, 77, 7520–7527. (22) Baeumner, A. J.; Pretz, J.; Fang, S. Anal. Chem. 2004, 76, 888–894. (23) Baeumner, A. J.; Schlesinger, N. A.; Slutzki, N. S.; Romano, J.; Lee, E. M.; Montagna, R. A. Anal. Chem. 2002, 74, 1442–1448. (24) Esch, M. B.; Baeumner, A. J.; Durst, R. A. Anal. Chem. 2001, 73, 3162– 3167. (25) Singh, A. K.; Harrison, S. H.; Schoeniger, J. S. Anal. Chem. 2000, 72, 6019– 6024. (26) Ho, J.-A. A.; Hsu, H.-W. Anal. Chem. 2003, 75, 4330–4334. (27) Ho, J.-A. A.; Wu, L.-C.; Huang, M.-R.; Lin, Y.-J.; Baeumner, A. J.; Durst, R. A. Anal. Chem. 2007, 79, 246–250. (28) Ho, J.-A. A.; Hung, C.-H. Anal. Chem. 2008, 80, 6405–6409. (29) Ho, J.-A. A.; Hung, C.-H.; Wu, L.-C.; Liao, M.-Y. Anal. Chem. 2009, 81, 5671–5677. (30) Ho, J.-A. A.; Huang, M.-R. Anal. Chem. 2005, 77, 3431–3436. (31) Viswanathan, S.; Wu, L.-C.; Huang, M.-R.; Ho, J.-A. A. Anal. Chem. 2006, 78, 1115–1121. (32) Liao, W.-C.; Ho, J.-A. A. Anal. Chem. 2009, 81, 2470–2476. (33) Zhan, W.; Bard, A. J. Anal. Chem. 2007, 79, 459–463. (34) Edwards, K. A.; Baeumner, A. J. Anal. Chem. 2007, 79, 1806–1815. (35) Mason, J. T.; Xu, L.; Sheng, Z.-M.; O’Leary, T. J. Nat. Biotechnol. 2006, 24, 555–557.

as a valuable platform to combine nucleic acid amplification with immunoassay for ultrasensitive protein detection. We have recently reported an aptamer-based immuno-RCA assay for protein detection.36 This technique is demonstrated to be able to avoid the conjugation of DNA with antibodies and offer a wide dynamic range with a desirably low detection limit. However, aptamers for most important protein biomarkers are currently unavailable, and like the DNA-antibody conjugates, aptamers are also exposed to the risk of electrostatic adsorption on proteins as well as chemical or enzymatic degradation. Herein, we reported for the first time a DNA encapsulating liposome based RCA immunoassay, liposome-RCA immunoassay, as an alternative strategy. In contrast to quantitative PCR that requires thermal cycling and real-time detection, RCA is an isothermal nucleic acid amplification technique and allows the detection at the end of reactions.37-43 This circumvents the need of costly instruments for temperature cycling and real-time detection, which makes this strategy compatible with point-of-care applications. Also, we introduce a biobarcode design44,45 in the liposome-entrapped DNA sequences. This enables the strategy to be immediately implemented for multiplex assay of multiple proteins in a cost-efficient manner. In the present study, we demonstrate that the liposomeRCA immunoassay can be exploited to determine a model protein target, prostate-specific antigen (PSA), a highly selective molecular biomarker associated with prostate cancer. The developed liposome-RCA immunoassay offers several salient advantages, including preparation of the DNA-carrying antibody with ease, protection of DNA labels from degradation by external impurities, low nonspecific adsorption of liposomes on protein components, ultrahigh sensitivity provided by primary amplification via releasing numerous DNA primers from liposomes followed by a secondary RCA amplification, and biobarcode-based multiplex assay for multiple protein targets. Therefore, it is expected that this strategy can offer a more versatile, sensitive, and robust platform to combine the nucleic acid amplification with immunoassay for ultrasensitive protein detection. EXPERIMENTAL SECTION Reagents and Apparatus. Human PSA, carcinoembryonic antigen (CEA), R-fetoprotein (AFP), immunoglobulin M (IgM), and immunoglobulin A (IgA) was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). PSA140 monoclonal antibody (mAb1) and PSA103 monoclonal antibody (mAb2) were obtained from Tianjian Biotechnology Co. Ltd. (Tianjin, China). Bovine serum albumin (BSA), human serum albumin (HSA), human immu(36) Zhou, L.; Ou, L.-J.; Chu, X.; Shen, G.-L.; Yu, R.-Q. Anal. Chem. 2007, 79, 7492–7500. (37) Yang, L.; Fung, C. W.; Cho, E. J.; Ellington, A. D. Anal. Chem. 2007, 79, 3320–3329. (38) Cho, E. J.; Yang, L.; Levy, M.; Ellington, A. D. J. Am. Chem. Soc. 2005, 127, 2022–2023. (39) Tian, Y.; He, Y.; Mao, C. ChemBioChem 2006, 7, 1862–1864. (40) Ali, M. M.; Li, Y. Angew. Chem., Int. Ed. 2009, 48, 1–5. (41) Li, J. S.; Zhong, W. W. Anal. Chem. 2007, 79, 9030–9038. (42) Deng, Z.; Tian, Y.; Lee, S.-H.; Ribbe, A. E.; Mao, C. Angew. Chem., Int. Ed. 2005, 44, 3582–3585. (43) Zhao, W.; Gao, Y.; Kandadai, S. A.; Brook, M. A.; Li, Y. Angew. Chem., Int. Ed. 2006, 45, 2409–2413. (44) Bao, Y. P.; Wei, T.-F.; Lefebvre, P. A.; An, H.; He, L.; Kunkel, G. T.; Muller, U. R. Anal. Chem. 2006, 78, 2055–2059. (45) Hill, H. D.; Vega, R. A.; Mirkin, C. A. Anal. Chem. 2007, 79, 9218–9223.

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Table 1. Synthesized Probes (5′f 3′) Used in Liposome-RCA Immunoassaya primer probe 1 ligation probe 2 padlock probe 3

GCT GAC AAT ACA TTA GAC CGA GAG CCA ACC ACA GTG AAG ATA TAG ATA ATA GAG CAA AAT GAG TAG AAG ATA TA dA PO3-TTG CTC TAT TAT CTA TAT TTT CTT CAC TGT GGT TGG CTC TCG GTC TCT TGT ACT TCC CTT CAT TAT ATC TTC TAC TCA TT capture probe 4 TAA TGT ATT GTC AGC TTTTT-Biotin detection probe 5 CTC TCG GTC TCT T-FITC primer probe 6 GTA ATA CGA CGA CTA GAC CGA GAG CCA ACC ACA GTG AAG a Complementary sequences are given in the same font style. d at the 3′ terminus of probe 2 is dideoxynucleotide modification. PO3 at the 5′ terminus of probe 3 is phosphorylation modification. Probe 6 has a barcode sequence different from that of probe 1.

noglobulin G (IgG), thrombin, bovine serum, and high-binding 96-well microtiter plates were from Dingguo Biotechnology Co. Ltd. (Beijing, China). Glutaraldehyde, cholesterol, Sephadex G-100, streptavidin-coupled agarose microbeads, 1,2-dipalmitoylsn-glycero-3-phosphatidylethanolamine (DPPE), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were purchased from Sigma Chemical Co. (St. Louis, MO). Escherichia coli DNA ligase and deoxyribonucleoside 5′-triphosphate mixture (dNTPs) were obtained from Takara Biotechnology Co., Ltd. (Dalian, China). Bst DNA polymerase (large fragment) was provided by New England Biolabs (Ipswich, MA). The oligonucleotide probes used in this work were synthesized from Takara Biotechnology Co. Ltd. (Dalian, China), as shown in Table 1. The solutions were prepared using ultrapure water, which was obtained through a Millipore Milli-Q water purification system (Billerica, MA) and had an electric resistance >18.3 MΩ. The serum samples were provided by Hunan Provincial Tumor Hospital (Changsha, China). A PSA chemiluminescent immunoassay (CLIA) kit was purchased from Autobio Co. Ltd. (Zhengzhou, China). Fluorescence measurements were performed at room temperature in a 200 µL quartz cuvette on a Fluorolog3 spectrofluorometer (Jobin Yvon). The excitation wavelength was 494 nm and the emission spectra were recorded from 505 to 570 nm with both excitation and emission slits of 5 nm. The peak intensities were obtained at 518 nm. Fluorescence imaging observations were performed at room temperature on an Olympus BX51 epifluorescence microscope equipped with an electron multiplied CCD (DV887, Andor Technology, Belfast, North Ireland). The FITC-3540B filter set (482/35 exciter, 536/40 emitter, 506 dichroic) was purchased from Semrock (Rochester, NY). Preparation of DNA-Encapsulating Liposomes. DNAencapsulating liposomes were prepared using the film hydration method.30,35,46,47 Briefly, DPPC, cholesterol, and DPPE (10:10:1 molar ratio, 30 mg in total) were dissolved in 4 mL mixture of chloroform and methanol (6:1 volume ratio) followed by sufficient sonication. The solution was then subjected to rotary evaporation under reduced pressure (0.09 MPa) to remove the solvents. This produced a thin lipid film on the inside wall of the round-bottom flask. Multilamellar DNA-encapsulating vesicles were obtained via hydration of film using 2 mL of phosphate-buffered saline (PBS: 10 mM phosphate buffer, pH 7.2, 100 mM NaCl) containing 200 µM of DNA primer probe 1 at 45 °C with the flask rotated vigorously until the lipid film was peeled off the inside wall. To (46) Chen, H.; Zheng, Y.; Jiang, J.-H.; Wu, H.-L.; Shen, G.-L.; Yu, R.-Q. Biosens. Bioelectron. 2008, 24, 684–689. (47) Chen, H.; Jiang, J.-H.; Li, Y.-F.; Deng, T.; Shen, G.-L.; Yu, R.-Q. Biosens. Bioelectron. 2007, 22, 993–999.

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obtain unilamellar vesicles, the multilamellar liposomes were subjected to a sonication treatment (10 cycles of 4 min on and 1 min off) in an ice-water bath using a probe-type sonicator (20 W). The resulting liposome suspension was centrifuged at 3000 rpm for 15 min to remove undispersed lipids and multilamellar vesicles. The unencapsulated DNA oligonucleotides were removed from the liposome suspension through a Sephadex G-100 column at room temperature followed by dialysis using a membrane with molecular weight cutoff of 20 000 against PBS (10 mM phosphate buffer, pH 7.2, 100 mM NaCl) for 24 h at 4 °C. The resulting DNAencapsulating liposome suspension (∼5 mg lipid mL-1) was stored at 4 °C for future use. The hydrodynamic size of liposomes was determined by dynamic light scattering with a Malvern Zetasizer 3000 HS particle size analyzer (Malvern Instruments, Worcs, UK). The phospholipid content of the liposomes was determined using Bartlett phosphorus assays according to the literature.34 Conjugation of Antibodies to DNA-Encapsulating Liposomes. Liposome conjugates with PSA140 mAb1 were prepared via the glutaraldehyde coupling method:46,47 In 3 mL of 2.5% glutaraldehyde solution, 2 mL of DNA-encapsulating liposomes (∼5 mg lipid mL-1) was added in drops under gentle stirring followed by the incubation for 1 h at 25 °C. Excess glutaraldehyde was removed by dialysis against 100 volumes of PBS (10 mM phosphate buffer, pH 7.2, 100 mM NaCl) for 12 h at 4 °C. Then, 1 mL of PSA140 mAb1 (1 mg mL-1 in PBS) was added under gentle stirring followed by the incubation for 1 h at 25 °C. In order to block excess aldehyde groups on the liposome surface, 1 mL of BSA (10 mg mL-1 in PBS) was added and reacted under gentle stirring for 1 h at 25 °C. The uncoupled PSA140 mAb1 and BSA were separated from the liposomes by Sephadex G-100 column. The resulting DNAencapsulating immunoliposomes were stored in PBS (10 mM phosphate buffer, pH 7.2, 100 mM NaCl) containing 3% (w/w) BSA at 4 °C until use. In order to eliminate the nonspecific adsorption of the DNAencapsulating immunoliposomes on the microtiter wells, liposomes without encapsulated DNA and antibody modification were used as the blocking reagent. This blocking liposome was synthesized using the aforementioned procedure for preparing the DNA-encapsulating liposomes, but 2 mL of PBS (10 mM phosphate buffer pH 7.2, 100 mM NaCl) was used in place of the DNA-containing PBS to hydrate the lipid film, and the conjugation of antibody to liposomes was not performed. Also, a control immunoliposome was prepared in which the entrapped DNA primer probe 1 was replaced by probe 6 using the aforementioned procedure. This control liposome was used for validating the

specificity of the liposome-RCA immunoassay. The blocking and control liposomes (∼5 mg lipid mL-1) were stored at 4 °C until use. Preparation of Circular DNA Templates, Capture-ProbeModified Microbeads, and Antibody-Coated Microtiter Wells. To 26 µL of hybridization buffer containing 40 mM Tris-HCl (pH 8.8), 20 mM KCl, 20 mM (NH4)2SO4, 4 mM MgSO4, and 0.2% Triton X-100 were added 3 µL of ligation probe 2 (30 µM) and 1 µL of padlock probe 3 (30 µM) followed by the incubation at 55 °C for 2 h. After the mixture cooled to room temperature, 5 µL of E. coli DNA ligase (60 U µL-1), 10 µL 0.05% BSA, 7 µL of 10× ligase buffer (300 mM Tris-HCl, pH 8.0, 40 mM MgCl2, 100 mM (NH4)2SO4, 12 mM EDTA, and 1 mM NAD+), and 48 µL of ultrapure water were added, and the mixture was incubated overnight at 16 °C. The ligase was inactivated by heating at 65 °C for 20 min. The resulting circular DNA template solution was stored at 4 °C until use. The microbeads modified with capture probe 4 were prepared by using the streptavidin-biotin bridge, as briefly described as follows: The streptavidin-coupled microbeads were first washed using 2 mL of PBS (10 mM phosphate buffer pH 7.2, 100 mM NaCl) three times with the aid of centrifugation and resuspended in PBS with a final concentration of 10 mg mL-1. In 500 µL of streptavidin-coupled microbead solution, 500 µL of biotinylated capture probe 4 (20 µM) was added and incubated for 1 h at 37 °C. The capture-probe-modified microbeads were washed using 2 mL of PBS three times with the aid of centrifugation. The sediment was collected and resuspended in 0.5 mL of PBS. The resulting capture-probe-modified microbead solution was stored at 4 °C for future use. The high-binding microtiter wells were coated with PSA capture antibody via incubation using 100 µL of PSA103 mAb2 (100 µg mL-1) in a coating buffer (0.2 M carbonate buffer, pH 9.6) overnight at 4 °C. Unbound PSA103 mAb2 was removed by discarding the supernatants followed by wash with 200 µL of PBS (10 mM phosphate buffer pH 7.2, 100 mM NaCl) three times. The wells were blocked by incubation with 100 µL of 3% (w/w) BSA for 1 h at 37 °C. Then, the wells were washed as mentioned above and dried under a nitrogen stream. The microtiter plates were stored at 4 °C until use. Liposome-RCA immunoassay. A 100-µL aliquot of PSA sample (concentration ranging from 0 to 10-9 g mL-1) in PBS (10 mM phosphate buffer pH 7.2, 100 mM NaCl) containing 3% (w/w) BSA was added to a microtiter well and incubated for 0.5 h at 37 °C. The sample solution was discarded, and the well was washed using 200 µL of PBS (10 mM phosphate buffer pH 7.2, 100 mM NaCl) containing 3% (w/w) BSA three times. Then, 40 µL of PBS (10 mM phosphate buffer pH 7.2, 100 mM NaCl) containing 3% (w/w) BSA, 50 µL of blocking liposomes (5 mg lipid mL-1), and 10 µL of DNA-encapsulating immunoliposomes (5 mg lipid mL-1) were dispensed in turn into the well, which was incubated for 1 h at 37 °C. The liposome mixture solution was removed, and the well was washed using 200 µL of PBS (10 mM phosphate buffer pH 7.2, 100 mM NaCl) containing 3% (w/w) BSA three times. Subsequently, 100 µL of Triton X-100 (10 mM) was added in the well, which was incubated for 5 min to lyse the bound liposomes. The 100 µL

of lysate was collected and transferred into a microcentrifuge tube. The DNA prime probe 1 released from the liposomes could be taken to initiate a linear RCA reaction, as described as follows: To the 100 µL of lysate containing DNA probe 1 were added 50 µL of circular DNA template solution, 20 µL of 10× ThermoPol reaction buffer [200 mM Tris-HCl, pH 8.8, 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4 and 1% Triton X-100], 20 µL of 2.5 mM/each dNTP mixture, and 10 µL of Bst DNA polymerase (large fragment, 8 U µL-1) in turn. The RCA reaction started at 65 °C for 3 h and finally terminated by heating at 80 °C for 10 min to inactivate the Bst polymerase. After the reaction mixture cooled to room temperature, 10 µL of capture-probemodified microbeads and 10 µL of FITC-labeled detection probe 5 (10 µM) were added, and the mixture incubated at 37 °C for 1 h. After centrifugation at 3000 rpm at 4 °C for 3 min, the sediment was washed three times with PBS (10 mM phosphate buffer pH 7.2, 100 mM NaCl) and resuspended in 200 µL of PBS for fluorescence detection. RESULTS AND DISCUSSION Design of Liposome-RCA Immunoassay. The liposome-RCA immunoassay utilizes a sandwiched immunoassay format with the DNA-encapsulating immunoliposomes as the detection reagent. The design of this technique is illustrated in Figure 1. PSA samples are introduced in the microtiter wells, where the PSA antigens are captured by the immobilized PSA103 mAb2. The captured PSA antigens then interact with the PSA140 mAb1-modifed liposomes with DNA primer probe 1 entrapped, rendering the PSA140 mAb1-modifed liposomes bound on the well surface. The liposomes are ruptured by surfactant treatment to release the DNA primer probes. The DNA prime probes are designed to have a biobarcode sequence for selective capture of the probes and a universal primer sequence to initiate the RCA reaction. Then, the DNA primer probes are annealed on the circular templates through the universal primer sequence and initiate a linear RCA reaction in the presence of Bst DNA polymerase and dNTPs. This produces single-stranded tandem repeated copies of the circular template. Each RCA product has a tandem repeated sequence complementary to the FITClabeled detection probe 5 and a biobarcode sequence at the 5′ terminus that is complementary to the capture probe 4. Thus, FITC-labeled detection probes are hybridized with the RCA products, which are then annealed on the capture probemodified microbeads. Finally, the fluorescence signals of the microbeads are detected, which can be used as a quantitative measure for the PSA concentration in the samples. As the first demonstration of incorporating RCA in immunoliposomes for ultrasensitive protein detection, there are indeed several novel advantages intrinsic in the liposome-RCA immunoassay. First, liposome-RCA immunoassay comprises two signal amplification routes, the high number of DNA primers entrapped in the liposome and the RCA reaction to generate long tandem repeated sequence for fluorescence detection. Combining the low nonspecific adsorption of liposomes, this technique is expected to show very high detection sensitivity. Second, we design the liposome-encapsulated DNA primer probe to have a biobarcode sequence and a universal primer sequence. This barcode sequence identifies the liposome-encapsulated primer probe specific to an Analytical Chemistry, Vol. 81, No. 23, December 1, 2009

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Figure 1. Schematic representation of liposome-RCA immunoassay. (A) A circular template is synthesized using a padlock probe and a ligation probe (dideoxynucleotide modification at 3′ terminus, red dot) via DNA ligase reaction. (B) The presence of PSA mediates the formation of a sandwiched immunocomplex with DNA-entrapped immunoliposomes on the well surface. The primer probes (biobarcode sequence, red segment; universal primer sequence, black segment) are then released from liposomes. (C) Primer probes initiate RCA to generate a long tandem repeated sequence, and (D) RCA products are hybridized with FITC-labeled detection probes and captured by microbeads for fluorescence assay.

individual protein target. Therefore, with different barcode sequences specifically designed for individual protein targets, the liposome-RCA immunoassay can be directly implemented for multiplex detection of multiple proteins. Combining the employed microtiter format, this strategy holds great promise as a highthroughput technology for protein detection. The universal primer sequence enables RCA reactions for different barcoded primer probes using an identical circular template. This reduces the cost of synthesizing multiple circular templates in multiplex detection of different proteins. Third, end-point detection and isothermal reaction in RCA circumvents the need of costly instruments for temperature cycling and real-time detection, which makes this strategy a valuable tool for point-of-care applications. Fourth, the DNA-encapsulating immunoliposomes can be prepared easily using well-established protocols for immunoliposomes. This simplified the synthesis and purification steps for obtaining the DNA-antibody conjugates. Fifth, the phospholipid bilayer of the liposome can isolate DNA labels from external enzymes and most chemical agents, which improves the stability of DNA labels against degradation by external impurities. 9668

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Besides the aforementioned basic scheme for liposome-RCA immunoassay, some remarks should be made to clarify the implementation of the technique. First, the ligation probe 2 for preparing the circular template is designed to have a dideoxynucleotide modification at the 3′ terminal. Therefore, the ligation probe cannot serve as the primer to initiate RCA in the absence of other primers. This simplified the preparation of the circular templates. Otherwise, extensive exonuclease treatment is required to remove the ligation probes and eliminate nonspecific RCA reaction primed by the ligation probes. Second, Bst polymerase (large fragment) is used instead of the commonly used Phi29 polymerase in RCA. This polymerase is lacking in 3′f5′ exonucleolytic activity. This can improve the specificity of RCA, because mismatch at the 3′ terminal between DNA sequences from external impurities and the circular template cannot initialize efficiently the RCA reaction. Also, this allows the 3′ dideoxynucleotide-modified ligation probes to be used directly in the RCA reaction without priming nonspecific extensions. Characterization of DNA-Encapsulating Liposome. The characteristic parameters of the DNA-entrapped liposomes were

Table 2. Characteristics of the Liposomes hydrodynamic size (diameter, nm) volume of liposome (µL) DNA concentration (M) number of encapsulated DNA reporters per liposome liposome concentration (number/mL) number of antibody molecules on the liposome surface

146 ± 8 1.6 × 10-12 2.0 × 10-4 169 3.7 × 1012 ∼870

summarized in Table 2. The hydrodynamic size (diameter) of the liposomes, reported on the basis of intensity distribution, was 146 ± 8 nm, as determined by dynamic light scattering technique with the Malvern Zetasizer particle size analyzer. After antibody conjugation, the hydrodynamic size (diameter) of the liposomes increased to 189 ± 11 nm. The average volume of a single liposome was calculated to be 1.6 × 10-12 µL. Assuming a bilayer thickness of 4 nm, the entrapped volume was calculated to be ∼1.4 × 10-12 µL. Given that the concentration of the entrapped DNA labels inside the liposomes was identical to that for the original solution used (200 µM), one could calculate that each liposome contained ∼169 DNA primer probes. On the basis of RCA assay of the primer probes released from lysed liposomes with reference to the calibration curve obtained for the standard solutions of primer probes (as shown in the next section), it was possible to calculate that the liposome concentration was ∼3.7 × 1012 liposomes/mL. Considering the average surface area of the DPPC, DPPE and cholesterol was 0.71, 0.41, and 0.19 nm2, respectively,48,49 it was estimated that ∼6900 molecules of DPPE were on the outer surface of each liposome.26 The estimation of antibody molecules on the outer surface of each liposome could be performed using BCA (bicinchoninic acid) protein analysis.50 Then, the number of antibodies on the outer surface of each liposome was determined to be ∼870, indicating that the conjugation efficiency between antibody and DPPE was ∼13%. The stability of the DNA-encapsulating liposomes during storage was monitored through RCA assay of the DNA primer probes before and after lysis of the liposomes. The corresponding concentrations of DNA probes were calculated with reference to the standard curve of unencapsulated probe, as shown in the next section. The leakage percents, expressed as a ratio of the probe amount found in external solution divided by the total probe amount after lysis, were determined to be 3.4% and 10.8%, respectively, after storage at 4 °C for 2 and 4 months. This indicated that the liposomes were stable and retained their DNA encapsulant for 2 months and that more than 2-month storage could lead to a significant leakage of the encapsulated DNA probes. Thus, for applications where the liposome-RCA immunoassay was used over 2 months, it was recommended to recalibrate the method using standards of the protein targets. Proof-of-Principle of Liposome-RCA Immunoassay. Since RCA is a linear amplification procedure, it will produce tandem repeated sequences in proportion to the amount of primer probes. However, the fluorescence protocol for detecting the RCA (48) Singh, A. K.; Kilpatrick, P. K.; Carbonell, R. G. Biotechnol. Prog. 1996, 12, 272–280. (49) Israelachvili, J. N.; Mitchell, D. J. Biochim. Biophys. Acta 1975, 389, 13– 19. (50) Fleiner, M.; Benzinger, P.; Fichert, T.; Massing, U. Bioconjugate Chem. 2001, 12, 470–475.

Figure 2. Fluorescent emission spectra obtained in RCA assay of the DNA primer with varying concentrations (10-15, 10-14, 10-13, 10-12, 10-11, 10-10, 10-9 and 10-8 M). The arrow indicates the increase of primer concentrations. Inset: Fluorescence intensity at 518 nm as a function of logarithmic primer concentration.

products involves two hybridization processes between the RCA products and detection probes as well as capture probes, which may make this RCA assay deviate from linear dependency. Therefore, before implementing the liposome-RCA immunoassay, the quantitative nature of the RCA assay for the primer probe was investigated. Figure 2 depicts the fluorescence emission spectra obtained in the RCA assay for varying concentrations of DNA primer. It was observed that the fluorescence peaks increased with increasing DNA primer concentration within the range from 1 fM to 10 nM. In logarithmic scales, the fluorescence intensity at maximal emission wavelength exhibited a linear correlation to the primer concentration through a 7-decade range from 1 fM to 10 nM, and a signal-to-background ratio as high as 120 was obtained. These results demonstrated that the fluorescence-based RCA assay of DNA primer was a very sensitive approach for quantitative detection of the DNA primer. The high signal-tobackground ratio also implied that the RCA assay could offer improved detection precision. So, we sought to combine this technology with the liposome immunoassay for protein detection through the encapsulation of sequence-specific DNA primers. Figure 3A depicts the fluorescent spectra obtained in the liposome-RCA immunoassay in the presence of 0.1 ng mL-1 PSA and 10 µg mL-1 BSA. One observed that in the presence of PSA target, a well-defined fluorescence emission peak was achieved with a maximum at 518 nm (curve a), a typical fluorescence emission spectrum for FITC labels. In contrast, no noticeable fluorescent emission peak was obtained in the absence of PSA, even in the presence of excess another protein, such as 10 µg mL-1 BSA commonly used as the control protein in immunoassay (curve b). These observations conveyed that the liposome-RCA immunoassay gave specific response to the PSA target and that nonspecific background response to coexisting proteins was desirably low. One also noticed that the background fluorescence intensity was ∼6.8 × 104 at 518 nm, which might result from the nonspecific adsorption of the immunoliposomes on the microtiter well and nonspecific adsorption of the FITC-labeled detection probes on the agarose microbeads. The fluorescence signal for 0.1 ng mL-1 PSA Analytical Chemistry, Vol. 81, No. 23, December 1, 2009

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Figure 3. (A) Fluorescent emission spectra obtained in liposome-RCA immunoassay of 0.1 ng mL-1 PSA (a, dashed line) and 10 µg mL-1 BSA (b, dotted line) and in the experiment with control liposomes encapsulated with probe 6 in place of probe 1 to detect 0.1 ng mL-1 PSA (c, solid line). (B) Agarose gel (0.7%) electrophoresis images obtained in liposome-RCA immunoassay of 10 µg mL-1 BSA (lanes 1 and 2) and 0.1 ng mL-1 PSA (lanes 3 and 4). Lane M: DNA size marker.

sample was 8.1 × 106 at 518 nm, which yielded an excellent signal-to-background ratio of ∼119. Note that this excellent signal-to-background ratio was also attributed to the use of both 3% (w/v) BSA and the blocking liposome (5-fold concentration of the liposome detection reagent) as the blocking reagents. It was found that only one blocking reagent such as BSA or the blocking liposome gave a background of 16.4 × 104 or 25.7 × 104, confirming the effectiveness of the use of dual blocking reagents. A further control experiment was performed by using the control liposome encapsulated with DNA probe 6 in place of the immunoliposome entrapped with primer probe 1 to detect 0.1 ng mL-1 PSA. Probe 6 was designed to be different from primer probe 1 in the barcode sequence. In this case, the fluorescence spectrum obtained (curve c) was very close to that achieved for the aforementioned blank sample. This finding revealed that the presented liposome-RCA immunoassay was very specific to the designed barcode sequence in the encapsulated DNA primer. This gave immediate evidence that the use of a unique barcode sequence for a protein target enabled specific detection of the protein without the interference from other DNA sequences encoded for other proteins, indicating the potential of the liposome-RCA immunoassay for multiplex detection of multiple protein targets. The liposome-RCA immunoassay was also verified using agarose gel electrophoresis experiments, as shown in Figure 3B. It was observed that no RCA products were obtained in the absence of PSA target, whereas the presence of 0.1 ng mL-1 PSA yielded RCA products with extremely low mobility, indicators of the large molecular weight of the RCA products and the high efficiency of RCA reaction. A further inspection of the liposome-RCA immunoassay was performed using fluorescence microscopy. Figure 4 shows the microscopic images of the agarose microbeads obtained in the liposome-RCA immunoassay. Because the microbeads had an average diameter of ∼5 µm, these microbeads could be visualized directly in the optical microscopic images either for the PSA sample or for the blank sample, as shown in Figure 4, parts A and B. In the fluorescence microscopic image shown in Figure 9670

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4C, no microbead was visible for the blank sample, suggesting that few detection probes were captured on the microbeads and that the RCA products were not produced in the assay. In contrast, the fluorescence microscopic image for the PSA sample shown in Figure 4D rendered all microbeads as bright and visible, implying that the RCA products were captured on the microbeads together with the FITC-labeled detection probes. These results further confirmed the potential of the liposome-RCA immunoassay for specific detection of protein targets. Optimization of Liposome-RCA Immunoassay. The fluorescence responses were dependent upon the amount of the FITClabeled detection probes bound to the RCA products. The greater the number of tandem repeated copies of the circular template present, the greater the amount of bound detection probe. So, a long RCA reaction time was expected to yield enhanced signal. Figure 5 depicts the effect of RCA reaction time on the fluorescence intensity. One observed that the fluorescence intensity increased rapidly with the RCA reaction time up to 3 h and became saturated over 3 h. Thus, 3 h was selected as the optimum time for the RCA reaction. The concentration of DNA-entrapped immunoliposome also had great effect on the fluorescence signal intensity. So, the influence of the concentration of the liposome detection reagent on the assay performance was investigated, as shown in Figure 6. It was observed that the fluorescence intensity increased with decreasing dilution ratios (increasing concentration) of the liposome detection reagent up to 1:10. There was no significant increase in the fluorescence signal as the dilution ratio became smaller than 1:10. To avoid possible nonspecific adsorption, the optimum dilution ratio of 1:10 was selected for the subsequent assay. The effects of other conditions on the assay performance were also investigated. One major difference in RCA reaction conditions for the liposome-RCA immunoassay was the introduction of 10 mM Triton X-100, which was commonly used to lyse the captured liposomes. So, the effect of 10 mM Triton X-100 on the efficiency of RCA reaction was examined. Fortunately, the presence of 10 mM Triton X-100 was found to

Figure 4. Optical microscopic images of agarose microbeads obtained in liposome-RCA immunoassay of 10 µg mL-1 BSA (A) and 0.1 ng mL-1 PSA (B). Fluorescence microscopic images of agarose microbeads obtained in liposome-RCA immunoassay of 10 µg mL-1 BSA (C) and 0.1 ng mL-1 PSA (D).

Figure 5. Effect of RCA reaction time on fluorescence response in liposome-RCA immunoassay. The results were the average of three repetitive experiments with error bars indicating the standard deviation.

Figure 6. Effect of dilution ratio of DNA-entrapped immunoliposomes on fluorescence response in liposome-RCA immunoassay. The results were the average of three repetitive experiments with error bars indicating the standard deviation.

have little influence on the efficiency of RCA reaction, which enabled the use of 10 mM Triton X-100 for liposome lysis in the liposome-RCA immunoassay. Assay Performance of Liposome-RCA Immunoassay. Figure 7A depicts the fluorescence emission spectra obtained in the liposome-RCA immunoassay in response to different PSA concentrations. One observed that the fluorescence intensity increased with increasing PSA concentration in the range from 0.1 fg mL-1 to 0.1 ng mL-1. Figure 7B depicts the calibration curve of the liposome-RCA immunoassay. On logarithmic scales, the fluorescence intensity at the maximum emission wavelength was found to exhibit a linear correlation to PSA concentration over a 6-decade concentration range from 0.1 fg mL-1 to 0.1 ng mL-1 with a linear correlation coefficient of 0.992. The dynamic range

was about 2-4 orders of magnitude wider than those shown previously.16,44 Furthermore, the dose response was linear over 2 orders of magnitude, indicating that the developed liposomeRCA immunoassay has a high dose-response sensitivity and was expected to show improved precision in PSA determination. A limit of detection as low as 0.08 fg mL-1 was estimated in terms of the 3σ rule. Such a detection limit (LOD) for PSA was 1-3 orders of magnitude lower than those reported by most sensitive assay methods currently in use for PSA detection, such as immuno-RCA (LOD of 0.1 pg mL-1),16 nanoparticlebased biobarcode technology (LOD of 1 fg mL-1),44 and siliconnanowire field-effect sensor (LOD of 0.9 pg mL-1).51 Such a high sensitivity of the liposome-RCA immunoassay was attributed to the double signal amplification of the liposome and Analytical Chemistry, Vol. 81, No. 23, December 1, 2009

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Figure 8. Fluorescence intensity responses of liposome-RCA immunoassay to PBS buffer, other proteins (IgG, 10 µg mL-1; IgM, 30 µg mL-1; IgA, 10 µg mL-1; HSA, 10 µg mL-1; CEA, 120 ng mL-1; thrombin 15 µg mL-1; AFP, 200 ng mL-1), and undiluted bovine serum in the absence or presence of 0.1 ng mL-1 PSA. The results were the average of three repetitive experiments with error bars indicating the standard deviation. Table 3. Comparison of Liposome-RCA Immunoassaya with the Chemiluminescent Immunoassay (CLIA)

Figure 7. (A) Fluorescence emission spectra obtained in liposomeRCA immunoassay of PSA with varying concentrations (10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 10-10, and 10-9 g mL-1). The arrow indicates the increase of PSA concentration. (B) Fluorescence intensity at 518 nm as a function of logarithmic PSA concentration. The results were the average of three repetitive experiments with error bars indicating the standard deviation.

the RCA assay as well as the low nonspecific adsorption of liposomes. Additionally, the largest value of coefficients of variation for triplicate measurements was 8.7%, indicating that this proposed method exhibited excellent reproducibility. The specificity of the liposome-RCA immunoassay was also examined using other proteins, such as IgG, IgM, IgA, HSA, CEA, thrombin, and AFP, commonly present in human serum. About 103-105-fold concentrations of these coexisting proteins were used in the assay. Also, to investigate the interference of complex matrices, bovine serum without dilution was assayed. The fluorescence responses of the developed liposome-RCA immunoassay to these proteins or matrix in the absence or presence of 0.1 ng mL-1 PSA are shown in Figure 8. It was observed that no significant fluorescence signals were obtained for these interfering proteins and matrix and the presence of these interfering proteins or matrix has little interference with the detection of PSA using the presented method. Thus, no significant cross-reactivity was detected for these proteins or matrix, implying high specificity of the liposome-RCA immunoassay for PSA detection. (51) Zheng, G.; Patolsky, F.; Cui, Y.; Wang, W. U.; Lieber, C. M. Nat. Biotechnol. 2005, 23, 1294–1301.

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sample

liposome-RCA immunoassay (ng mL-1)

CLIA (ng mL-1)

relative deviation (%)

1 2 3 4 5 6 7 8 9 10 11

87.37 ± 3.93 16.71 ± 0.93 37.95 ± 2.03 6.84 ± 0.58 11.04 ± 0.93 41.31 ± 2.33 6.62 ± 0.53 11.18 ± 0.86 73.50 ± 3.66 3.03 ± 0.28 5.88 ± 0.53

83.57 17.93 35.98 6.29 11.92 43.60 6.09 10.42 77.16 2.77 6.40

4.55 -6.80 5.48 8.74 -7.38 -5.25 8.70 7.29 -4.74 9.39 -8.13

a The data are given as the average ± SD obtained in three repetitive experiments.

The proposed method was further demonstrated for direct analysis of human serum specimens. Eleven human serum specimens from patients with prostate diseases were obtained from Hunan Provincial Tumor Hospital (Changsha, China) and determined by the liposome-RCA immunoassay as well as a commercialized CLIA kit as the reference. The results are shown in Table 3. One observed that the PSA concentrations obtained using the developed method were in good agreement with those determined by the commercial CLIA kit, and the relative deviations were not more than 9.39%. Such deviations were acceptable in common immunoassay practice, since these assays usually involved multistep reactions on a solid surface and the analyte concentration was typically below the ng mL-1 range. This suggested that it was feasible to apply the developed liposomeRCA immunoassay method to detecting PSA in human serum samples. CONCLUSIONS A novel liposome-RCA immunoassay technique was developed as a versatile platform to combine the RCA nucleic acid amplifica-

tion with immunoassay for ultrasensitive protein detection. This technique offered enormous signal amplification due to primary amplification via releasing numerous DNA primers from a liposome followed by a secondary RCA amplification. Combining the low nonspecific adsorption of liposomes, this technique was expected to show very high detection sensitivity. This technique also enabled easy preparation of the DNA-carrying antibody, favorable protection of DNA labels from degradation, and isothermal end-point detection with simple instrumentation. A biobarcode design was incorporated in the technique to identify the protein target. Therefore, using different immunoliposome-encapsulated primer probes with barcode sequences specifically designed for individual protein targets, the liposome-RCA immunoassay could be directly expanded for multiplex protein detection. It was

demonstrated that the technique was several orders of magnitude more sensitive than existing methods and gave high specificity and dose-response sensitivity. Combining the microtiter format, this strategy holds great promise as an ultrasensitive, specific, and multiplex technology for protein detection in proteomics and clinical diagnostics. ACKNOWLEDGMENT This work was supported by NSF (20975035) of China and “973” National Key Basic Research Program (2007CB310500). Received for review August 7, 2009. Accepted October 12, 2009. AC901786M

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