Exploration of Nanoparticle-Mediated Photothermal Effect of. Quantitative Photothermal Immunoassay
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1 Analytical Chemistry Supporting Information Exploration of Nanoparticle-Mediated Photothermal Effect of TMB-H 2 O 2 Colorimetric System and Its Application in a Visual Quantitative Photothermal Immunoassay Guanglei Fu, 1 Sharma T. Sanjay, 1 Wan Zhou, 1 Rolf A. Brekken, 2 Robert A. Kirken, 3 and XiuJun Li 1,4,5, * 1 Department of Chemistry and Biochemistry, University of Texas at El Paso, 500 West University Ave., El Paso, Texas, 79968, USA 2 Hamon Center for Therapeutic Oncology Research, Departments of Surgery and Pharmacology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, Texas, 75390, USA 3 Department of Biological Sciences, University of Texas at El Paso, 500 West University Ave., El Paso, Texas, 79968, USA 4 Biomedical Engineering, Border Biomedical Research Center, University of Texas at El Paso, 500 West University Ave., El Paso, Texas, 79968, USA 5 Environmental Science and Engineering, University of Texas at El Paso, 500 West *Corresponding author: xli4@utep.edu University Ave., El Paso, Texas, 79968, USA S-1
2 Table of Contents EXPERIMENTAL SECTION...S-3 Materials and Instruments...S-3 Preparation of Antibody-Conjugated Iron Oxide NPs......S-3 Photothermal Characterization of the Iron Oxide NPs-Mediated TMB-H 2 O 2 Colorimetric System... S-4 Iron Oxide NPs-Mediated TMB-H 2 O 2 Colorimetric Immunoassay... S-5 Specificity of the Iron Oxide NPs-Mediated TMB-H 2 O 2 Photothermal Immunoassay... S-6 RESULTS AND DISCUSSION...S-6 TEM Characterization of Formation of the Sandwich-Type Immunoassay...S-6 REFERENCES...S-7 S-2
3 EXPERIMENTAL SECTION Materials and Instruments. We purchased 3,3,5,5 -tetramethylbenzidine (TMB), prostate-specific antigen (PSA), horseradish peroxidase (HRP), bovine serum albumin (BSA), and serum from normal human (male AB plasma) from Sigma-Aldrich (Burlington, MA, USA). Iron oxide NPs (Carboxyl-functionalized; 30 nm in diameter) were obtained from Ocean NanoTech (San Diego, CA, USA). Hepatitis B surface antigen (HBsAg) was purchased from Fitzgerald Industries International (Acton, MA, USA). Carcino-embryonic antigen (CEA), monoclonal mouse anti-human PSA antibody and polyclonal rabbit anti-human PSA antibody were obtained from Abcam (Cambridge, MA, USA). The measurement of UV-Vis absorption spectra was carried out on a SPECTROstar Nano Microplate Reader (BMG LABTECH, Cary, NC, USA) utilizing a 96-well microplate. A pen-style digital thermometer (model number KT-300 LCD) with a detection range of -50 to +300 o C was obtained from a local supermarket. A diode laser with output power from 0 to 2.5 W was purchased from Opto Engine (Midvale, UT, USA; model number MDL-III-808; wavelength 808 nm; ~7 x 17 x 5 cm in dimension). Preparation of Antibody-Conjugated Iron Oxide NPs. Antibody-conjugated iron oxide nanoparticles (NPs) were prepared using the carbodiimide method according to the literature. 1 Briefly, 1.0 mg iron oxide NPs were suspended in 2.0 ml deionized water under ultrasonication, followed by the addition of 25.0 µl aqueous mixture of S-3
4 N-hydroxysulfosuccinimide (25 mg ml -1 ) and 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (25 mg ml -1 ) for further incubation at room temperature for 30 min. Afterwards, 80.0 µg polyclonal rabbit antihuman PSA antibody was added into the nanoparticle suspension under gentle stirring, followed by further reactions at room temperature for 2.0 h. To collect the antibody-conjugated nanoparticles, the nanoparticle suspension was centrifuged at rpm for 10 min at 4.0 o C. The obtained precipitates were washed with phosphate-buffered saline (PBS; ph = 7.4, 0.01 M) 3 times. Finally, the as-prepared antibody-conjugated iron oxide NPs were suspended in 2.0 ml PBS containing 0.2% BSA. Photothermal Characterization of the Iron Oxide NPs-Mediated TMB-H 2 O 2 Colorimetric System. To monitor the photothermal effect of the Fe 3 O 4 NPs-mediated TMB-H 2 O 2 colorimetric reaction solutions (without immunoassays involved), different concentrations of Fe 3 O 4 NPs were dispersed in 0.15 ml phosphate-citrate buffer solutions (0.2 M, ph = 5.0) containing TMB (0.4 mm) and H 2 O 2 (1.0 M) in a 96-well microplate. After incubation for 40 min, the reaction solutions (0.15 ml) were transferred into PCR tubes (0.2 ml), which were then irradiated vertically with an 808 nm laser for different amounts of time (10-60 s) at a power density of 5.26 W cm -2. A pen-style digital thermometer probe was immediately inserted into the solutions to monitor the temperature after the irradiation. The highest steady vachieved after insertion of the thermometer probe for about 5-8 s was recorded as the photothermal measurement signal. S-4
5 To monitor the photothermal process of the Fe 3 O 4 NPs-mediated TMB-H 2 O 2 colorimetric reaction solutions, the colorimetric reaction solutions (1.0 ml, mg ml -1 Fe 3 O 4 NPs) were then transferred into glass cuvettes (1.5 ml), which were then irradiated horizontally with the laser for 10 min at a power density of 3.12 W cm -2. A pen-style digital thermometer was inserted into the solutions to monitor the temperature during the irradiation process. Iron Oxide NPs-Mediated TMB-H 2 O 2 Colorimetric Immunoassay. A 120 µl monoclonal mouse anti-human PSA antibody solution (30.0 µg ml -1 ) was pipetted into each polymerase chain reaction (PCR) tube (200 µl), followed by incubation at 4.0 o C overnight. A 200 µl blocking buffer solution containing 5.0% BSA was then used to block each PCR tube. Afterwards, 120 µl normal human serum (3-fold diluted with phosphate-buffered saline) spiked with different concentrations of standard PSA was pipetted into each PCR tube for further incubation at 37.5 o C for 2.0 h. After thorough washing of the PCR tubes, a 120 µl polyclonal anti-psa antibody-conjugated iron oxide NPs dispersion (0.5 mg ml -1 ) was pipetted into each PCR tube for further incubation for 2.0 h at 37.5 o C. The PCR tubes were finally thoroughly washed with phosphate-buffered saline to conduct the Fe 3 O 4 NPs-mediated TMB-H 2 O 2 colorimetric immunoassay described below. To conduct the colorimetric immunoassay, a 150 µl phosphate-citrate buffer solution (0.2 M, ph = 5.0) containing TMB (0.4 mm) and H 2 O 2 (1.0 M) was added in each PCR tube, followed by incubation at room temperature for 40 min. The solutions S-5
6 were thoroughly mixed every 5 min during the incubation period. After incubation for 40 min, the colorimetric reaction solutions were used for photograph collection, UV-Vis absorption spectroscopic characterization, and the photothermal immunoassay. Specificity of the Iron Oxide NPs-Mediated TMB-H 2 O 2 Photothermal Immunoassay. To study the specificity of the developed Fe 3 O 4 NPs-mediated TMB-H 2 O 2 photothermal immunoassay, different interfering substances, including BSA, immunoglobulin G (IgG), HBsAg, and CEA were measured using both the Fe 3 O 4 NPs-mediated TMB-H 2 O 2 colorimetric immunoassay and the photothermal immunoassay. For this purpose of validation, normal human serum samples (3-fold diluted with PBS) spiked with BSA (320 ng ml -1 ), IgG (320 ng ml -1 ), HBsAg (320 ng ml -1 ), CEA (320 ng ml -1 ), and PSA (32.0 ng ml -1 ) were tested with the colorimetric, UV-Vis absorption spectroscopic, and photothermal immunoassays, respectively. RESULTS AND DISCUSSION TEM Characterization of Formation of the Sandwich-Type Immunoassay. Transmission electron microscopy (TEM) was used to characterize iron oxide NPs captured in the sandwich-type immunoassay system, as shown in Figure S-1. After thorough washing, spherical iron oxide NPs were still observed at the substrate surface by TEM after completing the sandwich-type immunoreactions (Figure S-1B), S-6
7 with consistent morphology with that from the manufacturer (Ocean NanoTech, San Diego, CA, USA; Figure S-1A), indirectly indicating the successful completion of the sandwich-type immunoassay and the successful conjugation of antibody onto iron oxide NPs. Otherwise, iron oxide NPs would have been washed away and not imaged by TEM anymore. Figure S-1. (A) Transmission electron microscopic images of iron oxide NPs (from Ocean NanoTech) before antibody conjugation and (B) after the completion of sandwich-type immunoreactions. References 1. Jaetao, J. E.; Butler, K. S.; Adolphi, N. L.; Lovato, D. M.; Bryant, H. C.; Rabinowitz, I.; Winter, S. S.; Tessier, T. E.; Hathaway, H. J.; Bergemann, C.; Flynn, E. R.; Larson, R. S. Enhanced leukemia cell detection using a novel magnetic needle and nanoparticles. Cancer Res. 2009, 69, S-7
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