Custom Conjugation Anti Troponin I Gold (40nm)


An easy, rapid and solution phase method to detect human cardiac troponin I have been described to detect myocardial damage using gold nano bar-based biosensors. Detection is demonstrated by the distinctive shift of the longitudinal surface plasmon resonance wavelength of gold nanorods to specific antibody and antigen-binding events. For increased sensitivity, the aspect ratio of the gold nanorods is increased to about 5.5 simply by adding a small amount of HCl in a seed-mediated growth solution. The experimental results show that the detection limit of the present method is 10 ng/ml. Contrast tests reveal that these gold nanorod-based plasmonic biosensors have much higher sensitivity than conventional spherical gold nanoparticles.

Experimental sections

1. Materials

Cetyltrimethylammonium bromide (CTAB, 99%, Cat #: H6269) and poly (styrene sulfonate) (PSS, Mw 70,000) were purchased from Sigma. Hydrogen tetrachloroauric acid (HAuCl4 · 4H2O, 99%), hydrochloric acid (HCl), ribbon nitrate, sodium borohydride (NaBH4, 96%) and L-ascorbic acid (AA) were purchased from Shanghai Chemical Reagent Co. Ltd (China). Millipore quality water (18.18 MΩ / cm) was used throughout the experiments. Human myocardial muscle cTnI and anti-human cTnI monoclonal antibodies used in the experiment were obtained from the Cardiovascular Diseases Research Institute of the First Affiliated Hospital of Nanjing Medical University.

2. Manufacture of monodisperse gold NRs

Gold NRs were manufactured based on a seed-mediated CTAB-assisted growth procedure by Nikoobakht et al. [20] with some modifications. The gold seed solution was first prepared by adding 0.6 ml of an ice-cold solution of 10 mM NaBH4 to 10 ml of 0.25 mM HAuCl4 prepared in 0.1M CTAB solution, with vigorous stirring for 2 min. The yellow colour immediately changed to brown, indicating the formation of gold seeds. These seeds were aged for 2 h to allow hydrolysis of the unreacted NaBH4.

The growth procedure was extended to obtain a 100 ml dispersion of the gold NRs. Briefly, the solutions were added to a 250 ml conical flask, in the following order: 100 mL of 0.1 M CTAB solution, 1.0 mL of 10 mM silver nitrate solution, 2 mL of 25 mM aqueous HAuCl4, and 0.4 mL of 1 M HCl solution. To this was added 0.70 ml of 0.0788 M AA as a reducing agent and the mixture was homogenized with gentle stirring. Finally, 120 μL of seed solution were added and the entire solution was left to stand overnight (14-16 h).

3. PSS coating

Typically 10 ml of gold NR was centrifuged as manufactured at 13,000 g / min for 20 min, the supernatant was discarded and the precipitate was redispersed in 5 ml of Milli-Q water. Subsequently, it was added dropwise to 1 ml of aqueous PSS solution (2.5 mg / L) with vigorous stirring. After adding the PSS solution, the mixture was allowed to stand for 0.5 h. Then, it was centrifuged twice at 12,500 g / min to remove excess polyelectrolyte and dispersed in 5 mL of Tris buffer (pH = 8.2).

4. Conjugation of anti-h-cTnI antibody with PSS capped gold NR

The PSS covered gold NRs were mixed with an excess amount of anti-h-cTnI solution (50 µg / ml in Tris buffer) for 30 min with magnetic stirring. The mixture was centrifuged to remove non-binding antibodies and redispersed in Tris buffer. Finally, these antibody-conjugated gold NRs were stored at 4 ° C for later use.

5. Binding of h-cTnI to anti-h-cTnI antibody-conjugated gold NR

In each experiment, 1.0 ml of anti-cTnI antibody conjugated gold NR was added to a fixed amount (1, 10, 100, 200, 400, 500 ng) of h-cTnI under vortex mixing. The resulting mixture was incubated for 15 min before recording with the 2802S spectrophotometer (UNICO).

6. Characterization

Transmission electron microscopy (TEM) images were taken with a JEM-2000EX (JEOL) transmission electron microscope operated at 120 kV. Samples were prepared by dropping the gold product dispersion onto the carbon-coated copper grid and room dried. UV-vis-NIR spectra were recorded on a 2802S spectrophotometer (UNICO) in the range 300-1,100 nm. Fourier transform infrared (FTIR) spectra were measured on a Magna FTIR-750 spectrometer (Nicolet) and the vacuum dried sample was prepared as a KBr pellet.

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