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Materials: Carboxylated single-walled CNT powder was purchased from Cheaptubes. Inc. PDMS (Sylgard 184) was purchased from Dow Corning, Inc. 6-Monoacetylmorphine (MAM) was purchased from Cerilliant Analytical Reference Standards. Sodium dodecyl sulfate (SDS), tween-20, poly(ethylene glycol) (PEG), skim milk, bovine serum albumin (BSA), complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), sodium dihydrogen phosphate (NaH 2PO4), and disodium hydrogen phosphate (Na 2HPO4) were purchased from Sigma Aldrich. 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC) and Nhydroxysulfosuccinimide (sulfo-NHS) were purchased from Pierce Chemicals. Protein-A Sepharose was procured from Amersham Biosciences, India. Biomolecule synthesis: Synthesis of the carboxylic acid derivative of monoacetylmorphine (MAM-COOH), its bioconjugation with BSA, and the generation of antimorphine antibodies was reported in an earlier study.[5] Briefly, for the synthesis of hapten, the derivatization was done by refluxing the reaction mixture for 3 h at 90 °C in an inert nitrogen atmosphere, containing 3 μM of MAM, 24 μM of chloroacetic acid, 45 μM sodium hydroxide, and 30 μM acetonitrile. The presence of -COOH group was confirmed by thin-layer chromatograph (TLC) and IR spectroscopy.[5] The derivatized hapten (MAM-COOH) was used for conjugation with BSA (carrier protein) using carbodiimide coupling chemistry. For the activation, 50 μM MAM-COOH, 75 μM EDC, and 75 μM sulfo-NHS were mixed and incubated for 1 h at room temperature, followed by overnight incubation at 4 °C, and centrifuged for 10 min at 10000 g to remove urea precipitate. For the conjugation of activated hapten with BSA, 30 μM, of activated hapten was mixed with 0.15 μM (10 mg) of BSA to prepare the molar ratio of 100:1. The antibodies were raised against MAM-BSA conjugate in sixto eight-week-old New Zealand white rabbits. The rabbits were immunized subcutaneously with 250 μg of MAM-BSA mixed with equal volume of CFA at the time of first booster followed by IFA in subsequent booster doses. The rabbits were bled after the fifth day of each booster and blood was collected, serum precipitated, and antibodies (IgG) purified using Protein A sepharose column. The fractions were then dialyzed against PBS, and IgG concentration was determined at 280 nm and stored at -20 °C until used. Monodisperse (30-nm) colloidal gold was prepared using a modified Frens method. A 200 mL solution of 0.01% tetrachloroauric acid in Milli-Q water was brought to boiling. 4mL sodium citrate solution (1% w/v) was added to the boiling gold chloride solution. The solution was allowed to boil for 10 min until it developed a typical bright wine-red color of colloidal gold. The average particles size of colloidal gold was determined using a TEM (Hitachi Model H-7500) operated at 120 kV. The average particle size was estimated to be approximately 30±4 nm. For the preparation of antibody gold conjugate, 90 μg of antimorphine antibody was prepared in 20mM phosphate buffer, pH 7.4, and added dropwise into 1mL colloidal gold solution ([Au]=2.4×10-4 mol L-1) under mild stirring. The pH of the colloidal gold solution was maintained at 7.4 by addition of 10mM Na 2CO3 before adding antibody. The mixture was incubated overnight at 4 °C and centrifuged at 12000 rpm for 30 min to remove unconjugated antibody from the solution. The pellet obtained was washed three times with 10mM Tris (pH 8.0) containing 3% BSA under centrifugation at 12000 rpm for 30 min to remove traces of unconjugated antibody. The pellet was resuspended in 2mL of phosphate buffer (20mM, pH 7.4) and stored at 4 °C before use. The final concentration of colloidal gold in the antibody-gold conjugate solution was 4.8×10-4 mol L-1. A Hitachi 2800 UV/vis spectrophotometer was used to measure the absorbance of gold nanoparticles and antibody-labeled gold nanoparticle Preparation of CNT suspension: To improve the carboxylic functional groups for better molecule attachment, 20 mg of aspurchased carboxylated CNT material was acid treated in 100 mL 3:1 volume ratio of concentrated H2SO4 and HNO 3 and refluxed overnight at 50 °C. The refluxed solution was centrifuged several times to separate the tubes from the acid solution and neutralize the solution until pH 6. To further enhance tube dispersity, the 1mgmL-1 treated solution was diluted 10 times with deionized water to 0.1 mg mL-1 concentration and 1 w/v% SDS was added into the final volume. The suspension was sonicated, followed by centrifugation for 1 h at 14000 rpm to remove the non-dispersed bundles. The extracted supernatant was kept as a stock solution for the entire experiment. Immunosensor preparation: The fabrication process of flexible CNT/PDMS LGFET is mentioned in the section 2 (Figure 1). In our earlier study on protein-CNT interaction,[27] carboxylated singlewalled CNTs were shown to interact better with BSA. Hence, the acid-treated carboxylated single-walled CNTs prepared using the above-mentioned treatment was used as the active channel of the LGFET. The carboxylic groups were first activated with EDC and sulfo-NHS for 1 h at room temperature, rinsed with 50mM PB solution (pH 9.5), followed by 10 μgmL-1 MAM-BSA injection and overnight incubation at 4 °C. Excess unbound molecules were removed by rinsing the microchannel with copious PB solution, and the device was ready for target antibody detection. Electrical measurement: Electrical measurement of the CNTLGFET was performed using a homemade LabView system with a testing protocol similar to that in the reported literature.[16,28] For real-time monitoring, a liquid-gate potential (Vg) at -0.5 V was applied to the electrolyte through a reference electrode (3M KCl, FLEXREF, World Precision Instruments) and a small drain bias (Vd) of 10mV applied over the source and drain electrodes to obtain the kinetic response at respective sensing steps.
- 出版日期2010-5-7
- 单位南阳理工学院
