Emplate for the detection and quantification of the corresponding analyte, thus the specific sequence is not critical [16]. In general, the template should be <100 base-pairs in length to maximize encapsulation into the liposomes and consist of a sequence not likely to be found in the samples being analyzed. For the CEA assay an 84-base segment derived from the human 2-microglobin transcript was used. This segment spans an intron and thus is unlikely to be present in human serum. The reporter was prepared by cloning 2-microglobin cDNA, prepared and amplified from HeLa cell RNA, into a pCR2.1-TOPO T/A plasmid vector, which was used to transform On-Shot E. Coli (Invitrogen). A detailed description of reporter preparation using this method [16] is given in additional file 1: Supplementary information, under the section entitled "Preparation of DNA reporters". The reporters used in the assay control studies,Liposomes were prepared by mixing chloroform solutions of DSPC (24.5 mol ), cholesterol (45 mol ), DODAP (25 mol ), DSPE-mPEG(2000) (4.75 mol- ), DSPE-PEG(2000)Biotin (0.25 mol ), and DHPErhodamine (0.5 mol ). The solvent containing the lipid mixture (25 mg total lipid) was evaporated by drying under a stream of N2, and then under high vacuum for at least 4 h. The dried lipid film was hydrated in 1 mL of 300 mM citrate buffer, pH 4, by vortexing the suspension at 70 . The resulting multilamellar vesicles were then subjected to five freeze/thaw cycles using liquid nitrogen and a water bath set to 70 . The liposomes were extruded 10 times through two stacked 0.1-micron polycarbonate membranes at 70 using a vesicle extruder, which led to the formation of unilamellar liposomes 100 nm in diameter. Ethanol was then slowly added to the rapidly vortexed liposome suspension until the final ethanol concentration was 40 by volume. The reporter DNA (300 g) was added to the liposomes, which were incubated at 40 for 1 h and then dialyzed against 2 L of the citrate buffer using a 2,000 Da MWCO Disposo Dialyzer. The preparation was then dialyzed against 2 L of 20 mM HEPES buffer, 145 mM NaCl, pH 7.5. Unencapsulated reporter DNA was removed by ion-exchange gel filtration on DEAE-Sepharose CL-6B (0.5 ml of gel/ mg total lipid) using the HEPES buffer [17]. Falsenegative control liposomes were prepared as described above, but with 5 mol DSPE-mPEG(2000) and no DSPE-PEG(2000)Biotin. The 81 base-pair reporter derived from the TMV 126 kDa coat protein sequence was encapsulated into the liposomes.Determination of total lipid and PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27689333 total reporter concentrationDHPE-rhodamine (0.5 mol ) was included in the liposomes to facilitate the determination of lipid concentration. A 25-L aliquot of the liposome buy Leupeptin (hemisulfate) solution was added to a test tube along with 1.5 mL of methanol and 20 L of 0.1 N NaOH [18]. A blank was similarly prepared using PBS. The absorbance of the solution was read at 560 nm (A560) in a 1-cm path-length cell after zeroing the spectrophotometer against the blank. The total lipid concentration of the liposome solution wasHe et al. Journal of Nanobiotechnology 2012, 10:26 http://www.jnanobiotechnology.com/content/10/1/Page 5 ofthen calculated as A560 x 130 mol/mL (A560 x 88 mg/ mL). A 25-l aliquot of the liposome solution was combined with 350 L of 1 M NaCl and 1.125 mL of chloroform/methanol (2:1, v/v). A blank was similarly prepared using 25 L of PBS. The solutions were vortexed and allowed to stand for 10 min then the upper aqueous phase of each so.