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Universal liposomes: preparation and usage for the detection of mRNA

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Abstract

Dye-encapsulating liposomes can serve as signaling reagents in biosensors and biochemical assays in place of enzymes or fluorophores. Detailed here is the use and preparation of streptavidin-coupled liposomes which offer a universal approach to biotinylated target detection. The universal approach provides two advantages, i.e. only one type of liposome is necessary despite varying target and probe sequences and the hybridization event can take place in the absence of potential steric hindrance occurring from liposomes directly conjugated to probes. One objective of this work was to optimize the one-step conjugation of SRB-encapsulating liposomes to streptavidin using EDC. Liposome, EDC, streptavidin concentrations, and reaction times were varied. The optimal coupling conditions were found to be an EDC:carboxylated lipid:streptavidin molar ratio of 600:120:1 and a reaction time of 15 min. The second goal was to utilize these liposomes in sandwich hybridization microtiter plate-based assays using biotinylated reported probes as biorecognition elements. The assay was optimized in terms of probe spacer length, probe concentration, liposome concentration, and streptavidin coverage. Subsequently, the optimized protocol was applied to the detection of DNA and RNA sequences. A detection limit of 1.7 pmol L−1 and an assay range spanning four orders of magnitude (5 pmol L−1−50 nmol L−1) with a coefficient of variation ≤5.8% was found for synthetic DNA. For synthetic RNA the LOQ was half that of synthetic DNA. A comparison was made to alkaline phosphatase-conjugated streptavidin for detection which yielded a limit of quantitation approximately 80 times higher than that for liposomes in the same system. Thus, liposomes and the optimized sandwich hybridization method are well suited for detecting single-stranded nucleic acid sequences and compares favorably to other sandwich hybridization schemes recently described in the literature. The assay was then used successfully for the clear detection of mRNA amplified by nucleic acid sequence-based amplification (NASBA) isolated from as little as one Cryptosporidium parvum oocyst. The detection of mRNA from oocysts isolated from various water sample types using immunomagnetic separation was also assessed. Finally, to prove the wider applicability and sensitivity of this universal method, RNA amplified from the atxA gene of Bacillus anthracis was detected when the input to the preceding NASBA reaction was as low as 1.2 pg. This highly sensitive liposome-based microtiter plate assay is therefore a platform technology allowing for high throughput and wide availability for routine clinical and environmental laboratory applications.

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Abbreviations

AP:

Alkaline phosphatase

BSA:

Bovine serum albumin

DIG:

Digoxin

DPPC:

1,2-Dipalmitoyl-sn-glycero-3-phosphocholine

DPPE:

1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine

DPPG:

1,2-Dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt

EDC:

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

EDTA:

Ethylenediamine tetraacetic acid

HEPES:

N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid

HPV:

Human papillomavirus

HSS:

HEPES-saline-sucrose

IMS:

Immunomagnetic separation

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

LNA:

Locked nucleic acid

MES:

2-(4-Morpholino)ethanesulfonic acid

NASBA:

Nucleic acid-based sequence amplification

LOQ:

Limit of quantitation

OG:

n-Octyl-β-d-glucopyranoside

PBS:

Phosphate-buffered saline

PCR:

Polymerase chain reaction

SRB:

Sulforhodamine B

StAv:

Streptavidin

TBS:

Tris buffered saline

References

  1. Marx J (2000) Science 289:1670–1672

    Article  CAS  Google Scholar 

  2. Ranki M, Virtanen M, Palva A, Laaksonen M, Pettersson R, Kaariainen L, Halonen P, Soderlund H (1983) Curr Top Microbiol Immunol 104:307–318

    CAS  Google Scholar 

  3. Parkkinen S, Mantyjarvi R, Syrjanen K, Ranki M (1986) J Med Virol 20:279–288

    Article  CAS  Google Scholar 

  4. Lee CY, Panicker G, Bej AK (2003) J Microbiol Methods 53:199–209

    Article  CAS  Google Scholar 

  5. Namimatsu T, Tsuna M, Imai Y, Futo S, Mitsuse S, Sakano T, Sato S (2000) J Vet Med Sci 62:615–619

    Article  CAS  Google Scholar 

  6. Palkovics L, Burgyan J, Balazs E (1994) Res Virol 145:387–392

    Article  CAS  Google Scholar 

  7. Baeumner AJ, Leonard B, McElwee J, Montagna RA (2004) Anal Bioanal Chem 380:15–23

    Article  CAS  Google Scholar 

  8. Ranki M, Palva A, Virtanen M, Laaksonen M, Soderlund H (1983) Gene 21:77–85

    Article  CAS  Google Scholar 

  9. Leskela T, Tilsala-Timisjarvi A, Kusnetsov J, Neubauer P, Breitenstein A (2005) J Microbiol Methods 62:167–179

    Article  CAS  Google Scholar 

  10. Zhang N, Appella DH (2007) J Am Chem Soc 129:8424–8425

    Article  CAS  Google Scholar 

  11. Edwards KA, Baeumner AJ (2006) Anal Chem 78:1958–1966

    Article  CAS  Google Scholar 

  12. Ishii JK, Ghosh SS (1993) Bioconjug Chem 4:34–41

    Article  CAS  Google Scholar 

  13. Casademont I, Bizet C, Chevrier D, Guesdon JL (2000) Mol Cell Probes 14:233–240

    Article  CAS  Google Scholar 

  14. Aubin JT, Boulay D, Chapus A, Brechot C, Laure F, Agut H (1995) Res Virol 146:75–79

    Article  CAS  Google Scholar 

  15. Li ZP, Liu CH, Fan YS, Duan XR (2007) Anal Bioanal Chem 387:613–618

    Article  CAS  Google Scholar 

  16. Rule GS, Montagna RA, Durst RA (1996) Clin Chem 42:1206–1209

    CAS  Google Scholar 

  17. Edwards KA, Baeumner AJ (2006) Anal Bioanal Chem 386:1335–1343

    Article  CAS  Google Scholar 

  18. Edwards KA, Baeumner AJ (2006) Talanta 68:1421–1431

    Article  CAS  Google Scholar 

  19. Rongen HAH, Bult A, vanBennekom WP (1997) J Immunol Methods 204:105–133

    Article  CAS  Google Scholar 

  20. Locascio LE, Hong JS, Gaitan M (2002) Electrophoresis 23:799–804

    Article  CAS  Google Scholar 

  21. Park JW, Kirpotin DB, Hong K, Shalaby R, Shao Y, Nielsen UB, Marks JD, Papahadjopoulos D, Benz CC (2001) J Controlled Release 74:95–113

    Article  CAS  Google Scholar 

  22. Pardridge WM (2007) Adv Drug Deliv Rev

  23. Edwards KA, Baeumner AJ (2007) Anal Chem 79:1806–1815

    Article  CAS  Google Scholar 

  24. Campbell CH, Miller AL, Rome LH (1983) Biochem J 214:413–419

    CAS  Google Scholar 

  25. Madhankumar AB, Slagle-Webb B, Mintz A, Sheehan JM, Connor JR (2006) Mol Cancer Ther 5:3162–3169

    Article  CAS  Google Scholar 

  26. Willis MC, Collins BD, Zhang T, Green LS, Sebesta DP, Bell C, Kellogg E, Gill SC, Magallanez A, Knauer S, Bendele RA, Gill PS, Janjic N (1998) Bioconjug Chem 9:573–582

    Article  CAS  Google Scholar 

  27. Edwards KA, March JC (2007) Anal Biochem 368:39–48

    Article  CAS  Google Scholar 

  28. Ahn-Yoon S, DeCory TR, Baeumner AJ, Durst RA (2003) Anal Chem 75:2256–2261

    Article  CAS  Google Scholar 

  29. Ahn-Yoon S, DeCory TR, Durst RA (2004) Anal Bioanal Chem 378:68–75

    Article  CAS  Google Scholar 

  30. Hutchinson FJ, Jones MN (1988) FEBS Lett 234:493–496

    Article  CAS  Google Scholar 

  31. Liautard JP, Vidal M, Philippot JR (1985) Cell Biol Int Rep 9:1123–1137

    Article  CAS  Google Scholar 

  32. Duzgunes N, Pretzer E, Simoes S, Slepushkin V, Konopka K, Flasher D, de Lima MC (1999) Mol Membr Biol 16:111–118

    Article  CAS  Google Scholar 

  33. Chen C-S, Baeumner A, Durst R (2005) Talanta 67:205–211

    Article  CAS  Google Scholar 

  34. Plant AL, Brizgys MV, Locasio-Brown L, Durst RA (1989) Anal Biochem 176:420–426

    Article  CAS  Google Scholar 

  35. Baeumner AJ, Jones C, Wong CY, Price A (2004) Anal Bioanal Chem 378:1587–1593

    Article  CAS  Google Scholar 

  36. Wen HW, DeCory TR, Borejsza-Wysocki W, Durst RA (2006) Talanta 68:1264–1272

    Article  CAS  Google Scholar 

  37. Weber PC, Ohlendorf DH, Wendoloski JJ, Salemme FR (1989) Science 243:85–88

    Article  CAS  Google Scholar 

  38. Martin FJ, Papahadjopoulos D (1982) J Biol Chem 257:286–288

    CAS  Google Scholar 

  39. Martin FJ, Hubbell WL, Papahadjopoulos D (1981) Biochemistry 20:4229–4238

    Article  CAS  Google Scholar 

  40. Weissig V, Lasch J, Klibanov AL, Torchilin VP (1986) FEBS Lett 202:86–90

    Article  CAS  Google Scholar 

  41. Bendas G, Vogel J, Bakowski U, Krause A, Muller J, Rothe U (1997) Biochim Biophys Acta 1325:297–308

    Article  CAS  Google Scholar 

  42. Bredehorst R, Ligler FS, Kusterbeck AW, Chang EL, Gaber BP, Vogel CW (1986) Biochemistry 25:5693–5698

    Article  CAS  Google Scholar 

  43. MacKenzie WR, Hoxie NJ, Proctor ME, Gradus MS, Blair KA, Peterson DE, Kazmierczak JJ, Addiss DG, Fox KR, Rose JB et al (1994) N Engl J Med 331:161–167

    Article  CAS  Google Scholar 

  44. Sischo WM, Atwill ER, Lanyon LE, George J (2000) Prev Vet Med 43:253–267

    Article  CAS  Google Scholar 

  45. Fayer R, Trout JM, Lewis EJ, Santin M, Zhou L, Lal AA, **ao L (2003) Parasitol Res 89:141–145

    CAS  Google Scholar 

  46. Nime FA, Burek JD, Page DL, Holscher MA, Yardley JH (1976) Gastroenterology 70:592–598

    CAS  Google Scholar 

  47. Dragon DC, Bader DE, Mitchell J, Woollen N (2005) Appl Environ Microbiol 71:1610–1615

    Article  CAS  Google Scholar 

  48. Dahlgren CM, Buchanan LM, Decker HM, Freed SW, Phillips CR, Brachman PS (1960) Am J Hyg 72:24–31

    CAS  Google Scholar 

  49. Sanderson WT, Stoddard RR, Echt AS, Piacitelli CA, Kim D, Horan J, Davies MM, McCleery RE, Muller P, Schnorr TM, Ward EM, Hales TR (2004) J Appl Microbiol 96:1048–1056

    Article  CAS  Google Scholar 

  50. Cook N (2003) J Microbiol Methods 53:165–174

    Article  CAS  Google Scholar 

  51. Keer JT, Birch L (2003) J Microbiol Methods 53:175–183

    Article  CAS  Google Scholar 

  52. Koppel DE (1972) J Chem Phys 57:4814

    Article  CAS  Google Scholar 

  53. Frisken BJ (2001) Appl Optics 40:4087–4091

    Article  CAS  Google Scholar 

  54. Connelly J, Nugen S, Borejsza-Wysocki W, Durst R, Montagna R, Baeumner A (2008) Anal Bioanal Chem

  55. Bartlett GR (1959) J Biol Chem 234:466–468

    CAS  Google Scholar 

  56. Fiske CH, Subbarow Y (1925) J Biol Chem 66:375–400

    CAS  Google Scholar 

  57. Singh AK, Kilpatrick PK, Carbonell RG (1996) Biotechnol Prog 12:272–280

    Article  CAS  Google Scholar 

  58. Ege C, Lee KY (2004) Biophys J 87:1732–1740

    Article  CAS  Google Scholar 

  59. Israelachvili JN, Mitchell DJ (1975) Biochim Biophys Acta 389:13–19

    Article  CAS  Google Scholar 

  60. Hartley HA, Baeumner AJ (2003) Anal Bioanal Chem 376:319–327

    CAS  Google Scholar 

  61. Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J (1990) J Clin Microbiol 28:495–503

    CAS  Google Scholar 

  62. Gottschalk PG, Dunn JR (2005) Anal Biochem 343:54–65

    Article  CAS  Google Scholar 

  63. Gottschalk PG, Dunn JR, Vol Tech note 3022 Bio-Rad Laboratories Inc, Hercules CA USA

  64. Thompson V, Schatzlein D, Mercuro D (2003) Spectroscopy 18:112–114

    Google Scholar 

  65. Edwards KA, Baeumner AJ (2006) Anal Bioanal Chem 386:1613–1623

    Article  CAS  Google Scholar 

  66. Grabarek Z, Gergely J (1990) Anal Biochem 185:131–135

    Article  CAS  Google Scholar 

  67. Bogdanov AA Jr, Klibanov AL, Torchilin VP (1988) FEBS Lett 231:381–384

    Article  CAS  Google Scholar 

  68. Kohsaka H, Taniguchi A, Richman DD, Carson DA (1993) Nucleic Acids Res 21:3469–3472

    Article  CAS  Google Scholar 

  69. Running JA, Urdea MS (1990) Biotechniques 8:276–279

    Article  CAS  Google Scholar 

  70. Nickerson DA, Kaiser R, Lappin S, Stewart J, Hood L, Landegren U (1990) Proc Natl Acad Sci USA 87:8923–8927

    Article  CAS  Google Scholar 

  71. Jeltsch A, Fritz A, Alves J, Wolfes H, **oud A (1993) Anal Biochem 213:234–240

    Article  CAS  Google Scholar 

  72. Yang B, Viscidi R, Yolken R (1993) Anal Biochem 213:422–425

    Article  CAS  Google Scholar 

  73. Nikiforov TT, Rogers YH (1995) Anal Biochem 227:201–209

    Article  CAS  Google Scholar 

  74. Nikiforov TT, Rendle RB, Goelet P, Rogers YH, Kotewicz ML, Anderson S, Trainor GL, Knapp MR (1994) Nucleic Acids Res 22:4167–4175

    Article  CAS  Google Scholar 

  75. Nikiforov TT, Rendle RB, Kotewicz ML, Rogers YH (1994) PCR Methods Appl 3:285–291

    CAS  Google Scholar 

  76. Nunc (2007) Vol 2007 Nalge Nunc International

  77. Dai Z, Sirard JC, Mock M, Koehler TM (1995) Mol Microbiol 16:1171–1181

    Article  CAS  Google Scholar 

  78. Baeumner AJ, Humiston M, Montagna RA, Durst RA (2001) Anal Chem 73:1176–1180

    Article  CAS  Google Scholar 

  79. Esch MB, Baeumner AJ, Durst RA (2001) Anal Chem 73:3162–3167

    Article  CAS  Google Scholar 

  80. Rautio J, Barken KB, Lahdenpera J, Breitenstein A, Molin S, Neubauer P (2003) Microb Cell Fact 2:4

    Article  Google Scholar 

  81. Chen Y, Chi Y, Wen H, Lu Z (2007) Anal Chem 79:960–965

    Article  CAS  Google Scholar 

  82. Goral VN, Zaytseva NV, Baeumner AJ (2006) Lab Chip 6:414–421

    Article  CAS  Google Scholar 

  83. Wicks B, Cook DB, Barer MR, O’Donnell AG, Self CH (1998) Anal Biochem 259:258–264

    Article  CAS  Google Scholar 

  84. Nugen SR, Leonard B, Baeumner AJ (2007) Biosens Bioelectron 22:2442–2448

    Article  CAS  Google Scholar 

  85. Clavel C, Masure M, Levert M, Putaud I, Mangeonjean C, Lorenzato M, Nazeyrollas P, Gabriel R, Quereux C, Birembaut P (2000) Diagn Mol Pathol 9:145–150

    Article  CAS  Google Scholar 

  86. Neely LA, Patel S, Garver J, Gallo M, Hackett M, McLaughlin S, Nadel M, Harris J, Gullans S, Rooke J (2006) Nat Methods 3:41–46

    Article  CAS  Google Scholar 

  87. Kauppinen S, Vester B, Wengel J (2006) Handb Exp Pharmacol:405–422

Download references

Acknowledgements

The authors are grateful to Ravi Sood for his preliminary investigations into optimizations of microtiter plate-based DNA hybridization methodology. John Connelly provided the C. parvum NASBA amplicons from environmental water samples for this work. This project was funded in part by the CD4 Initiative, Imperial College, London, UK.

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  1. Biological and Environmental Engineering, Cornell University, 145 Riley-Robb Hall, Ithaca, New York, 14853, USA

    Katie A. Edwards, Katherine L. Curtis, Jessica L. Sailor & Antje J. Baeumner

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  1. Katie A. Edwards
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  2. Katherine L. Curtis
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Correspondence to Antje J. Baeumner.

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Edwards, K.A., Curtis, K.L., Sailor, J.L. et al. Universal liposomes: preparation and usage for the detection of mRNA. Anal Bioanal Chem 391, 1689–1702 (2008). https://doi.org/10.1007/s00216-008-1992-1

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