3.2.1 Preparation of Starch-Au NP complex
A 5.0 mg/mL stock solution of starch (Soluble Starch Extrapure; SRL) was prepared by dissolving 800.0 mg of starch in 160.0 mL boiling water. NP synthesis was carried out by adding 160.0 µL of 1.73 X 10-2 M HAuCl4 and 800.0 µL of H2O2 to the starch solution followed by ultrasonication for 20 min. The solution was left overnight and then centrifuged for 20 min at 20,000 rpm and at 250C. The supernatant was discarded and the pellet was resuspended in 10.0 mM phosphate buffer (pH 7.0). The cycle of centrifugation and buffer wash was performed twice more. Finally, the pellet was
suspended in 20.0 mL of buffer. The presence of Au NPs and starch in the solution was tested by UV-vis spectroscopy (using a Perkin Elmer Lambda25 spectrophotometer) and iodine test, respectively. Transmission electron microscopy (TEM) was performed using a Jeol 2100 machine (operating at 200 kV). The images were recorded after drop-coating solutions on carbon coated copper grids. All the subsequent experiments were performed with this solution only.
3.2.2 Estimation of starch in the composite using GOD-POD Method
150.0 mL of starch-Au NP solution (prepared as discussed in the above section) was centrifuged and redispersed in 20.0 mL buffer (pH 7.0). To 10.0 mL of this solution 100.0 µL of α-amylase (1.0 mg/mL) was added and incubated overnight at 370C, followed by centrifugation at 20,000 rpm to remove the Au NPs from it. The pH of the supernatant obtained was adjusted to 4.5, using dilute HCl, to which 30.0 µL of 3.0 mg / mL solution of amyloglucosidase (from Aspergillus niger, Fluka) was added and the solution was incubated at 550C for 8 h. The resultant mixture was tested for glucose using the glucose test kit based on GOD-POD method (GOD-POD Kit, Span Diagnostics, India). The amount of starch in the starch-Au NP composite was estimated by measuring the amount of glucose produced and comparing the same as obtained from known concentrations of starch solutions (0.03 mg/mL, 0.05 mg/mL and 0.5 mg/mL) under the same reaction conditions.
3.2.3 Enzymatic Starch Digestion Studies
For the starch digestion kinetics studies, 17.0 µL of 1.0 mg / mL α-amylase (from hog pancreas; Fluka), prepared in 10.0 mM phosphate buffer, was added to 26.0 mL of the starch-Au NP composite solution having a starch concentration of 0.05 mg / mL (as determined using GOD-POD method). An equivalent concentration of free starch solution was also taken for comparing kinetics of digestion of free starch vis-à-vis starch- Au NP composite. 2.0 mL aliquots from each of the reaction mixtures were withdrawn at regular time intervals; to each of which 100.0 µL of iodine solution (Gram’s Iodine, HiMedia) was added and the UV-vis spectra of the solutions were subsequently recorded (using a Perkin Elmer Lambda25 UV/Vis Spectrometer or Varian Cary 50 Bio UV-vis Spectrophotometer). For kinetics data the areas of the curves were plotted as a function of time.
- 48 - 3.2.4 Analysis of the enzyme digested starch-Au NP composite
a) Analysis of the presence of starch digested product(s): 60.0 µL of 1.0 mg / mL α- amylase solution was added to 10.0 mL of the starch-Au NP composite (so that the final concentration of protein is 0.14 µg / mL) and incubated overnight at 37°C. The solution was then centrifuged at 20,000 rpm for 20 min, followed by buffer wash of the precipitate. The precipitate was dispersed in 10.0 mL acetate buffer (pH 4.5) and the pH of the supernatant was adjusted to 4.5 using dilute HCl. Both the dispersed solution (from the precipitate) and the supernatant were then treated with 30.0 µL of 3.0 mg / mL solution of amyloglucosidase (from Aspergillus niger, Fluka), prepared in acetate buffer of pH 4.5, followed by incubation at 550C for 8 h. The solutions were then tested for the presence of glucose using the glucose test kit (GOD-POD Method, Span Diagnostics, India).
b) Analysis of the presence of enzyme: 1.4 mL of 1.0 mg/mL α-amylase solution was added to 13.6 mL of the starch-Au NP solution and centrifuged at 20,000 rpm for 20 min; the precipitate was then washed with buffer and the supernatant collected. The precipitate was resuspended in 1.5 mL buffer. Both the precipitate and the supernatant were tested for the presence of enzyme by the standard Bradford test for protein.20
3.2.5 Quantitative analysis for protein content after digestion of starch-Au NP composite
For this purpose, 1.0 mL each of the three enzyme (native α-amylase, native AMG and denatured α-amylase) solutions was added to 6.0 mL of starch-Au NP composite (separately) and all the reaction mixtures were kept at room temperature for 5 h. The solutions were then centrifuged at 20,000 rpm for 20 min, followed by the buffer wash of the precipitate obtained. The precipitate was redispersed in 1.5 mL buffer and the supernatant was also collected for analysis. Standard Bradford test for protein was conducted for the supernatant as well as the original stock solution of the enzyme used in the experiment. The amount of protein in the precipitate was calculated by the following equation.
Amount of protein in precipitate (Pp) = Amount of protein in 1 mL of the parent enzyme (PEnz) – Amount of protein in supernatant (Ps)
Also, iodine tests were performed with the precipitate and the supernatant for the presence of undigested starch, if any.
3.2.6 Electrophoretic detection of αααα-amylase-Au NP composite following digestion of starch-Au NP composite.
The digestion of starch-Au NP composite by α-amylase and subsequent recovery of the precipitate following digestion was accomplished as mentioned before. The presence of α-amylase in the pellet was ascertained by agarose (1.2%) gel electrophoresis.21 The samples analyzed were: starch-Au NP composite, α-amylase, α-amylase-Au NP composite obtained after digestion and Au NPs only. The samples were mixed in a ratio of 5:1 (v/v) with 30% glycerol and loaded on the gel. The conventional sample loading dye consisting of 30% glycerol and the tracking dyes bromophenol blue and xylene cyanol was loaded in one of the wells to monitor the electrophoretic run. Following electrophoresis, the gel was photographed and subsequently stained overnight with 0.1%
Coomasie Brilliant Blue R-250 prepared in a mixture of methanol and acetic acid (25:10 v/v). The gel was extensively destained in a solution of methanol and acetic acid (25:10 v/v) and the protein bands obtained were photographed appropriately.
3.2.7 Experiment for SPR based kinetics study
UV-vis spectra of the enzyme treated starch-Au NP composite were recorded at various time intervals. The areas under the curves were calculated and then plotted as functions of time. The iodine test for the digestion of starch in the composite was followed as before.