Anal. Chem. 2007, 79, 1806-1815
DNA-Oligonucleotide Encapsulating Liposomes as a Secondary Signal Amplification Means Katie A. Edwards and Antje J. Baeumner*
Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York 14853
A novel liposome-based signal amplification system was developed by encapsulating DNA oligonucleotides within antibody-tagged liposomes and subsequently detecting the oligonucleotide with dye-encapsulating liposomes for double signal enhancement. In this sandwich immunoassay, the model analyte, protective antigen protein from B. anthracis, was captured by one set of antibodies immobilized in microtiter plate wells and detected using a second antibody conjugated to oligonucleotide-encapsulating liposomes. Bound liposomes were lysed releasing the encapsulated fluorescein-tagged DNA 25-mer probe, which was then permitted to hybridize with its complementary sequence immobilized in a second plate. Finally, the amount of oligonucleotide was detected through the addition of anti-fluorescein antibody tagged dye-encapsulating liposomes. These secondary liposomes allowed for a ∼400× lower LOD than detection of the fluoresceinlabeled probe alone. Several aspects were investigated, including the encapsulation of various oligonucleotide concentrations within liposomes; oligonucleotide hybridization times and buffers; degree of anti-fluorescein antibody coverage on the liposomes; and immobilized anti-protective antigen antibody concentration. We found that the encapsulation efficiency increased with the starting oligonucleotide concentration. As many as 4000 DNA 25-mers were successfully entrapped in the liposome, and minimal leakage was observed over the course of 8 months. When used in the sandwich immunoassay, a limit of detection of 4.1 ng/mL protective antigen was observed with an upper limit of 5000 ng/mL. Due to the endless combination of DNA oligonucleotide sequences, this assay lends itself perfectly for multiplexing on the order of tens to hundreds of analytes. The success of immunoassays based on sandwich complex formation with antibody-labeled dye-encapsulating liposomes has been demonstrated for the detection of Escherichia coli,1 botulinum toxin,2 and cholera toxin.3 In these assays, the amount of signal due to the dye from the liposomes was proportional to the amount * Corresponding author. Tel.: +1-607-255-5433. Fax: +1-607-255-4080. E-mail:
[email protected]. (1) Park, S.; Durst, R. A. Anal. Biochem. 2000, 280, 151-158. (2) Ahn-Yoon, S.; DeCory, T. R.; Durst, R. A. Anal. Bioanal. Chem. 2004, 378, 68-75. (3) Ahn-Yoon, S.; DeCory, T. R.; Baeumner, A. J.; Durst, R. A. Anal. Chem. 2003, 75, 2256-2261.
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of analyte that participated in a sandwich complex. The benefit of dye-encapsulating liposomes over single fluorophore detection has been demonstrated previously, yielding a 500-1000-fold signal enhancement.4,5 We sought to combine this technology with the potential for a lower limit of detection and multiplexed analyses through the encapsulation of sequence-specific secondary target molecules. Toward this end, we encapsulated fluorescein-tagged DNA oligonucleotides within liposomes tagged with an antibody against protective antigen from Bacillus anthracis. Entrapment of nucleic acids within liposomes has been reported for antisense oligonucleotides,6,7 plasmids,8-10 for use as an internal control for real-time PCR,11 and reagents have been entrapped in liposomes to allow for internal DNA transcription12 and replication.13 The extent of encapsulation depends on several factors, including liposome preparation process, diameter, buffer, and lipid composition.14 Specifically, the salt concentration, composition, and lipid charge have been found to be important factors in maximizing the encapsulation of oligonucleotides.7,15 Several methods have been used to prepare nucleic acid incorporating liposomes, including the reverse-phase evaporation, freeze-thaw, and dehydration-rehydration methods. In this work, we compared HEPES to Tris buffers using the reverse-phase evaporation process for the encapsulation of a 100-600 µM solution of a fluoresceinlabeled 25-mer oligonucleotide. This primary population of antibody-tagged liposomes encapsulated between ∼300 and ∼4000 oligonucleotide molecules per liposome, depending on the initial probe concentration and resulting liposome diameter. Upon lysis of those liposomes participating in the sandwich immunoassay, the released fluoresceintagged oligonucleotide was then added to a second microtiter plate (4) Edwards, K., Baeumner, A. Anal. Bioanal. Chem. 2006, 386, 1613-1623. (5) Ho, J. A. A.; Durst, R. A. Anal. Biochem. 2003, 312, 7-13. (6) Semple, S. C.; Klimuk, S. K.; Harasym, T. O.; Dos Santos, N.; Ansell, S. M.; Wong, K. F.; Maurer, N.; Stark, H.; Cullis, P. R.; Hope, M. J.; Scherrer, P. Biochim. Biophys. Acta 2001, 1510, 152-166. (7) Fillion, P.; Desjardins, A.; Sayasith, K.; Lagace, J. Biochim. Biophys. Acta: Biomembr. 2001, 1515, 44-54. (8) Jeffs, L. B.; Palmer, L. R.; Ambegia, E. G.; Giesbrecht, C.; Ewanick, S.; MacLachlan, I. Pharm. Res. 2005, 22, 362-372. (9) Monnard, P. A.; Oberholzer, T.; Luisi, P. Biochim. Biophys. Acta 1997, 1329, 39-50. (10) Bailey, A. L.; Sullivan, S. M. Biochim. Biophys. Acta 2000, 1468, 239-252. (11) Berg, E. S.; Skaug, K. J. Microbiol Methods 2003, 55, 303-309. (12) Tsumoto, K.; Nomura, S.; Nakatani, Y.; Yoshikawa, K. Langmuir 2001, 17, 7225-7228. (13) Oberholzer, T.; Albrizio, M.; Luisi, P. L. Chem. Biol. 1995, 2, 677-682. (14) Kulkarni, S. B.; Betageri, G. V.; Singh, M. J. Microencapsulation 1995, 12, 229-246. (15) Lakkaraju, A.; Dubinsky, J. M.; Low, W. C.; Rahman, Y. E. J. Biol. Chem. 2001, 276, 32000-32007. 10.1021/ac061471s CCC: $37.00
© 2007 American Chemical Society Published on Web 01/25/2007
Figure 1. Schematic of assay. (A) Antibody-tagged DNA-encapsulating (primary) liposomes participate in sandwich immunoassay for protective antigen from B. anthracis. (B) Bound liposomes are lysed with surfactant to release the encapsulated fluorescein-labeled probe, which then (C) hybridizes with complementary sequence immobilized in a second microtiter plate. (D) Anti-fluorescein-tagged sulforhodamine B-encapsulating (secondary) liposomes bind to (E), fluorescein label on hybridized probe, and then (F) bound secondary liposomes are lysed with surfactant to yield a fluorescence signal proportional to the original analyte concentration. Table 1. Oligonucleotide Sequences probe name
sequence (5′-3′)
labeled target unlabeled target biotinylated capture probe
fluorescein-CTg gCA gCA gCC ACT ggA TCT CTA A CTg gCA gCA gCC ACT ggA TCT CTA A biotin-TTAgAgATCCAgTggCTgCTgCCAg
in which a complementary sequence was immobilized. A second population of anti-fluorescein, antibody-tagged liposomes entrapping a dye was added binding to all of the hybridized DNA oligonucleotides. These liposomes were lysed and the amount of dye was detected and found to be proportional to the initial analyte concentration. The format of the assay is outlined in Figure 1. MATERIALS AND METHODS 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], sodium salt (DPPG), N-glutaryl 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE), and the extrusion membranes were purchased from Avanti Polar Lipids (Alabaster, AL). Sulforhodamine B (SRB) was purchased from Molecular Probes, Inc. (Eugene, OR) All other reagents used in these experiments were purchased from VWR (Bridgeport, NJ) The DNA sequences listed in Table 1 were purchased from Operon Biotechnologies, Inc. (Alameda, CA) or provided by Invitrogen Corp. The liposome size distribution was determined by dynamic light scattering with a DynaPro LSR (Proterion Corp., Piscattaway, NJ) using the Dynamics (version 6.3.01) software program and the Cumulants method of analysis.16,17 Fluorescence measurements were made using a FLX800 microtiter plate reader (BioTek Instruments, Winooski, VT). Optical density measurements (16) Koppel, D. E. J. Chem. Phys. 1972, 57, 4814-&. (17) Frisken, B. J. Appl. Opt. 2001, 40, 4087-4091.
were made at 532 nm in a Beckman DU220 spectrophotometer (Beckman Coulter, Fullerton, CA) by diluting 5 µL of liposome samples into 995 µL of 10 mM HEPES, 200 mM sodium chloride, 0.2 M sucrose, 0.01% sodium azide at pH 7.0 in a 1.5-mL spectrometer cell. Liposome Preparation. SRB-Encapsulating Liposome Preparation. SRB-encapsulating liposomes were prepared as previously described.18 Briefly, DPPC, DPPG, cholesterol, and N-glutaryl DPPE (40.3:21:51.7:7.3 µmol, respectively) were first dissolved in a solvent mixture containing 3 mL of chloroform, 0.5 mL of methanol, and 3 mL of isopropyl ether and sonicated to ensure homogeneous mixing. A 45 °C solution of dye (2 mL of sulforhodamine B, 150 mM in 0.2 M HEPES) was added to the lipid mixture while sonicating for a total of 4 min. The mixture was then placed onto the rotary evaporator, and the solvent was removed at 45 °C. The mixture was then transiently vortexed preceding and following an additional introduction of 2 mL of 45 °C 150 mM SRB. The mixture was then returned to the rotary evaporator before being extruded at 60 °C 21 times through 2.0-µm membranes, followed by 21 times through 0.6-µm membranes. The liposomes were then passed through a 20 × 1.7 cm column packed with Sephadex G-50 at ∼4 mL/min using 1× HEPES-saline-sucrose buffer (1× HSS: 10 mM HEPES, 200 mM sodium chloride, 0.01% sodium azide at pH 7.0, osmolality adjusted with sucrose (∼0.22 M) to 550 mmol/kg). The liposome (18) Hartley, H. A.; Baeumner, A. J. Anal. Bioanal. Chem. 2003, 376, 319-327.
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fractions containing high liposome densities were then combined and dialyzed overnight against the 1× HSS buffer. DNA-Encapsulating Liposome Preparation. DNA oligonucleotide-encapsulating liposomes were prepared by the standard reverse-phase evaporation procedure as described above, but instead of SRB, a 100-600 µM solution of fluorescein-labeled probe in either 1× HEPES-saline (10 mM HEPES, 200 mM sodium chloride, 0.01% sodium azide at pH 7.0) or 1× Tris-buffered saline (1× TBS: 20 mM Tris, 150 mM sodium chloride, 0.01% sodium azide at pH 7.0) was used as an encapsulant. Liposomes were also prepared using 50-200 µM solutions of the unlabeled probe in 1× TBS. Upon completion of evaporation, the liposomes were extruded 21× through 2.0-µm membranes and then half of the volume was subsequently passed 21× through 0.6-µm membranes. The liposomes were then subjected to separation using a column packed with Sepharose CL-4B and either 1× HSS or 1× Tris buffered saline-sucrose. Dialysis of liposomes was completed for 12 h against the buffer used for size exclusion chromatography at pH 9.1 and then 12 h against the same buffer at pH 7.5 Bartlett Phosphorus Assays. Liposomes were assayed for their phospholipid content using Bartlett assays,19 performed as follows: Liposomes (3 × 20 µL) were dehydrated at 180 °C for 10 min and then digested to inorganic phosphates with 0.5 mL of 3.33 N H2SO4 for 2 h at the same temperature. A 100-µL aliquot of 30% hydrogen peroxide was added to each sample, and the mixtures were returned to the oven for 1.5 h. The tubes were permitted to cool to ambient temperature prior to, and vortexed vigorously following each addition. Last, 4.6 mL of 0.22% ammonium molybdate and 0.2 mL of the Fiske-Subbarow reagent were added. The Fiske-Subbarow reagent was prepared by mixing 40 mL 15% (w/v) of sodium bisulfite, 0.2 g of sodium sulfite, and 0.1 g of 1-amino-4-naptholsulfonic acid at ambient temperature for 30 min and then filtering out undissolved solids. The tubes were then heated in a boiling water bath for 7 min and then quickly cooled in an ice-water bath. The absorbance at 830 nm was recorded. Standards prepared from potassium phosphate dibasic in deionized water were subjected concurrently to the same procedure. The phospholipid content of the liposomes was determined from a calibration curve prepared from the standards analyzed in each run. Values were corrected for encapsulated probe content, and the total lipid concentration was calculated by multiplying the phospholipid concentration by the initial ratio of total lipid to phospholipid. Lamellarity Determination. Liposomes were prepared using the reverse-phase evaporation process with 0.25 mol % 7-nitro-2,1,3benzodiazol-4-yl-dipalmitoylphosphatidylethanolamine (NBD-DPPE), in addition to the lipids used in all other experiments using 100 µM 25-mer probe without the fluorescein label and 1× TBS as the encapsulant. Using the phospholipid concentrations obtained from Bartlett assays, the liposomes were diluted to a phospholipid concentration of 10 µM and final volume of 2 mL with the osmolality-adjusted Tris-saline-sucrose buffer. The fluorescence was recorded prior to and after the addition of 20 µL of a freshly prepared 1 M sodium dithionite/1 M Tris, pH 10 solution using λex ) 490 nm and λem ) 528 nm, as described by McIntyre and Sleight.20 The addition of sodium dithionite to the formed lipo(19) Bartlett, G. R. J. Biol. Chem. 1959, 234, 466-468. (20) McIntyre, J. C.; Sleight, R. G. Biochemistry 1991, 30, 11819-11827.
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somes quenches the fluorescence of the fraction of NBD-DPPE, which is exposed to the outer medium. Equations for determining the theoretical fraction of lipid molecules exposed to the external aqueous phase were used as presented in a recent report by Matsuzaki et al.21 Conjugation of Antibodies to Liposomes. SRB-Encapsulating Liposomes to Anti-Fluorescein Antibody. To 75 µL of SRBencapsulating liposomes (15.04 mM total lipid) was added 0.01, 0.03, 0.05, 0.07, 0.1, or 0.15 mol % anti-fluorescein antibody. The volumes were equalized using 1× PBS. Then, 0.86 µL of 1-ethyl3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC; 100 mg/mL in 0.1 M MES, pH 4.6), was added and the reaction was placed on a shaker for 15 min. The reaction was then subjected to a size-exclusion chromatography column packed with Sepharose CL-4B. The fractions containing high concentrations of liposomes were retained for use in the assays. These mol % values are based on the total lipid concentration determined from the Bartlett assays; thus, actual percentages of coverage are roughly double since approximately half of the lipids available for coupling are available on the external face. DNA-Encapsulating Liposomes to Antiprotective Antigen Antibody. To 100 µL of 600 µM fluorescein-labeled DNA-encapsulating liposomes (26.02 mM total lipid) was added 0.05 mol % antiprotective antigen antibody. A 1.5-µL aliquot of EDC (100 mg/mL in 0.1 M MES, pH 4.6, 5 equiv) was added, and the reaction was placed on a shaker for 15 min. The reaction was then subjected to a size-exclusion chromatography column packed with Sepharose CL-4B. The fractions containing high concentrations of liposomes were retained for use in the assays. Calculation of DNA Probe Encapsulation Efficiency. The encapsulation efficiencies, reported as the millimole of DNA per mole of phospholipid, were determined by dividing HPLC-derived probe content by the phospholipid content from the Bartlett assays. The amount of probe released upon lysis with 50 mM n-octyl β-Dglucopyranoside (OG) was calculated based on a standard curve using unencapsulated probe. The HPLC methodology was 1.5 mL/ min. 1× HSS on a 4000SWXL column (Tosohaas) with detection at 260 nm. The percent encapsulation was expressed as a ratio of the final probe to lipid ratio divided by the initial probe to lipid ratio.
([DNA]final/[phospholipid]final) % encapsulation ) 100 ([DNA]initial/[phospholipid]initial) (1)
While typically expressed as the percentage of solute recovered versus that initially added, this approach accounts for incomplete recovery of phospholipids. Since highly concentrated liposomes were desirable for their utilization in the sandwich immunoassays, only those liposomes were collected and retained; thus calculation on the basis of input versus output would underestimate the probe recovery. DNA-Encapsulating Liposome Stability Measurements. Two batches of liposomes (100 and 250 µM fluorescein-labeled probe in HEPES buffer, 100 µL each) were dialyzed for 24 h in separate (21) Matsuzaki, K.; Murase, O.; Sugishita, K.; Yoneyama, S.; Akada, K.; Ueha, M.; Nakamura, A.; Kobayashi, S. Biochim. Biophys. Acta 2000, 1467, 219226.
50-kDa MWCO chambers against 1× HSS at pH 7.2, 8.2, and 9.2. Immediately after removing samples from dialysis, 50 µL of the retentates and 250 µL of 1× HSS pH 7.0 were mixed in Microcon YM-50 devices (Millipore, Burlington, MA) and centrifuged for 10 min at 1000 rpm. The filtrates (2 µL) were then analyzed using fluorescence read at λex ) 490 nm and λem ) 528 nm for their DNA content. The intact dialyzed liposomes were also analyzed using fluorescence. Long-term stability measurements were made by incubating fluorescein-labeled DNA-encapsulating liposomes in a microtiter plate coated with the sequence complementary to the encapsulant and then detecting the amount of fluoresceinlabeled probe found bound after washing the wells. The wells of this black Reacti-bind Neutravidin plate (Pierce, Rockford, IL) were first incubated with 100 µL of the biotinylated complementary sequence (0.1 µM in 1× phosphate-buffered saline (1× PBS; 10 mM potassium phosphate, 150 mM sodium chloride, 0.01% (w/ v) sodium azide) for 30 min and then washed with 3 × 200 µL 1× PBS. DNA-encapsulating liposomes (100 µL in 1× HSS) were added, and the plate was incubated for 30 min. The wells of the plate were then washed with 3 × 200 µL of 1× HSS, and the fluorescence signal of the hybridized external probe was read at λex ) 490 nm and λem ) 528 nm. A standard curve was prepared from the fluorescein-labeled probe alone using the same conditions. Calculation of Theoretical Number of Liposomes, Probe Molecules, and Antibodies. The number of lipids per liposome (Ntot) was calculated as shown in eq 2, where d is the hydrodynamic diameter from light scattering measurements, t is the bilayer thickness, and aL is the average headgroup surface area per lipid:22
Ntot ) (π/aL)[d2 + (d - 2t)2]
(2)
The bilayer thickness was assumed to be 40 Å and aL was calculated using values of 71, 45, and 19 Å2 for phosphatidylcholine, phosphatidylglycerol, and cholesterol, respectively.23,24 Using these values and weighting by the mole fraction of each component, the aL obtained for these liposomes was 47.9 Å2/lipid. The number of liposomes was then calculated by dividing the total lipid concentration by Ntot. The number of antibody molecules per liposome was calculated by dividing the number of antibody molecules per liter (taken from the initial total lipid input and the mol % tag) by the liposome concentration. A 100% reaction efficiency was assumed, and this number was doubled to account for the coupling reaction occurring only on the exterior face. The number of probe molecules per liposome was estimated based on the diameter and size distribution obtained using dynamic light scattering and the equation of a sphere to determine the volume of the inner cavity. A unilamellar population of spherical liposomes with a bilayer thickness of 40 Å that encapsulate oligonucleotides at the same concentration as the initial concentration of probe was assumed to be present for this analysis. Optimization of Encapsulated Probe Detection. General Procedure. The microtiter plate preparation was completed as described (22) Singh, A. K.; Kilpatrick, P. K.; Carbonell, R. G. Biotechnol. Prog. 1996, 12, 272-280. (23) Ege, C.; Lee, K. Y. Biophys. J. 2004, 87, 1732-1740. (24) Israelachvili, J. N.; Mitchell, D. J. Biochim. Biophys. Acta 1975, 389, 13-19.
in the liposome long-term stability section. Fluorescein-labeled probe (100 µL) was added to the complementary sequence-tagged plate and hybridized for 30 min. Anti-fluorescein antibody-tagged SRB-encapsulating liposomes (100 µL) were then added and incubated for 30 min. Unbound liposomes were then removed, and the wells were washed with 3 × 200 µL of HEPES-salinesucrose buffer. The wells were then tapped dry and remaining liposomes were lysed with 50 µL of 30 mM OG for 5 min. The fluorescence of the lysed liposomes participating in the complex was then read at λex ) 540 nm and λem ) 590 nm. The following variations were investigated: (a) hybridization of the fluoresceinlabeled probe (1, 10, 100, 500 nM) for times ranging from 5 to 90 min; (b) hybridization of the fluorescein-labeled probe (35 nM) in buffers composed of 0, 3, 6, and 9× sodium saline citrate buffer (SSC) with 0, 5, 15, and 30% (v/v) formamide (90 µL) with 30 µL of 30 mM OG; (c) liposomal antibody coverage ranging from 0.03 to 0.15 mol % of the total lipid input and liposome concentration normalized to an OD532 nm ) 0.196 ( 0.007 using 0-100 nM fluorescein-labeled probe for a target; (d) liposomes conjugated at 0.1 mol % antibody diluted to OD532 nm ranging from 0.14 to 0.65 in 0.2 M sucrose, 10 mM HEPES, mM sodium chloride, and 0.01% sodium azide) using 0-100 nM fluorescein-labeled probe for a target. Sandwich Immunoassays. The steps of the sandwich immunoassay are summarized in Table 2. Neutravidin-linked black microtiter plates preblocked with SuperBlock were washed according to the manufacturer’s (Pierce) instructions before use, which entailed the introduction and removal of 3 × 200 µL of 0.05% (w/v) Tween-20, 0.01% (w/v) BSA, in 20 mM Tris buffer (wash buffer). A 100-µL aliquot of 5, 7, 8.2, 10, and 13 µg/mL biotinylated antiprotective antigen antibody in wash buffer was added in triplicate to wells. Between 6 and 11 biotin molecules were present per antibody. Antibody solutions were permitted to incubate for 2 h. Unbound antibody was then removed by discarding the supernatants, and then the plates were tapped gently onto three layered Kimwipes (Kimberly-Clark, Roswell, GA) to remove any residual volume. The wells were washed with 3 × 200 µL of wash buffer and tapped dry following the same procedure. A 100-µL aliquot of protective antigen (concentrations ranging from 0 to 10 µg/mL in wash buffer) was then added and incubated for 30 min. Unbound antigen was then removed, and the wells were washed with 2 × 200 µL of wash buffer, followed by 100 µL of 1× HSS. DNA-encapsulating liposomes conjugated to antiprotective antigen antibodies were then added and incubated for 30 min. Unbound liposomes were removed and wells washed with 2 × 200 µL of 1× HSS buffer and tapped dry. The bound liposomes were then lysed with 50 µL of 30 mM OG using a 5-min incubation period. The 30-µL aliquots of the OG-containing supernatants were then transferred to a Neutravidin-coated microtiter plate to which had been immobilized the biotinylated complementary sequence at a concentration of 0.1 µM, which contained 70 µL of hybridization buffer (9× SSC, 30% (v/v) formamide, and 0.2% (w/v) Ficoll) per well. After hybridization for 30 min, the unbound probe was removed and wells were washed with 2 × 200 µL of hybridization buffer, followed by 200 µL of 1× HSS. The fluorescence of the hybridized probe participating in the complex was then read at λex ) 490 nm and λem ) 528 nm. The 100-µL aliquots of anti-fluorescein antibody-tagged SRB-encapsulating liposomes Analytical Chemistry, Vol. 79, No. 5, March 1, 2007
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Table 2. Sandwich Immunoassay Method Detailsa plate 1 steps
volume (mL)
no. of additions
200 100 200 100 200 200 100
3 1 3 1 2 1 1
wash buffer wash buffer wash buffer wash buffer wash buffer 1× HSS 1× HSS
200 50
2 1
1× HSS 30 mM OG
200 100 200 70 30 200 200 100
3 1 3 1 1 2 1 1
wash buffer 1× PBS wash buffer hybridization buffer 30 mM OG hybridization buffer 1× HSS 1× HSS
200 50
2 1
1× HSS 30 mM OG
prewash neutravidinylated plate apply biotinylated antiprotective antigen antibody remove unbound biotinylated antibodies introduce protective antigen remove unbound protective antigen remove unbound protective antigen introduce antiprotective antigen tagged DNAencapsulating liposomes remove unbound liposomes add n-octyl β-D-glucopyranoside to lyse bound liposomes read plate at λex ) 490 nm λem ) 528 nm plate 2 steps prewash neutravidinylated plate introduce biotinylated capture probe remove unbound biotinylated probe add hybridization buffer to wells of plate add lysed DNA-encapsulating liposomes from plate 1 remove unbound probes remove unbound probes add anti-fluorescein antibody-tagged SRBencapsulating liposomes remove unbound liposomes add n-octyl β-D-glucopyranoside to lyse bound liposomes read plate at λex ) 540 nm λem ) 590 nm
solutiona
incubation time (min) 120 30 30 5
30 30 30 5
a 1× HSS ) 10 mM HEPES, 200 mM sodium chloride, 0.01% sodium azide at pH 7.0; 1× PBS ) 10 mM potassium phosphate, 150 mM sodium chloride, 0.01% (w/v) sodium azide; hybridization buffer, 9× SSC, 30% (v/v) formamide, and 0.2% (w/v) Ficoll; wash buffer, 0.05% (w/v) Tween-20, 0.01% (w/v) BSA, in 20 mM Tris buffer.
(antibody coverage 0.05 mol % of the total lipid) diluted in 1× HSS were then added, and the resultant mixture was incubated for 30 min. Unbound liposomes were then removed, and the wells were washed with 2 × 200 µL of 1× HSS. The wells were then tapped dry, and remaining liposomes were lysed with 50 µL of 30 mM OG for 5 min. The fluorescence of the lysed liposomes participating in the complex was then read at λex ) 540/35 nm and λem ) 590/20 nm. The data were fit using a four-parameter logistic (eq 3) using XLFit software (IDBS, Bridgewater, NJ):
y)b+
a-b (1 + (x/c)d)
(3)
where a is the response at zero concentration, b is the response at maximum concentration, x is the protective antigen concentration, c is the concentration yielding 50% response, and d is the slope factor. The limit of detection throughout was defined as the background signal plus 3× its standard deviation. RESULTS AND DISCUSSION The first aspect of this work was to optimize the encapsulation of a 25-mer DNA oligonucleotide within liposomes using probe concentrations ranging from 50 to 600 µM with the reverse-phase evaporation process. The resulting liposome mixtures were extruded through either 2.0-µm pore size membranes only or extruded through 2.0-µm pore size membranes followed by 0.6-µm pore size membranes. Following extrusion, unencapsulated probe was separated from probe-encapsulating liposomes using size-exclusion chromatography. Clear baseline separation between liposomes and unencapsulated probe was observed using HPLC to monitor the absorbance of the fractions from the SEC column 1810
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at λ ) 260 nm. The first peak at an elution volume of 10-17 mL corresponded to DNA-encapsulating liposomes, whereas the peak from 24 to 33 mL corresponded to unencapsulated DNA oligonucleotides. Despite this clear separation by size, we found a minimal amount of probe (∼
