Supplementary Material
Fluorogenic Assays for Activated Protein C Using Aptamer Modified Magnetic Beads
Qiang Zhao1*, Jie Gao1,2
1. Research Center for Environmental Science and Engineering, Shanxi University, Taiyuan, China, 030006
2. College of Chemistry and Chemical Engineering, Shanxi University, Taiyuan, China, 030006
* Corresponding author, Email: , Tel: +86-351-7018525; Fax: +86-351-7011011
Preparation of the aptamer modified magnetic beads
Streptavidin-coated magnetic beads (100 μL, 10 mg.mL-1) were added into a tube and separated from the solution by using a magnetic separator. The collected beads were washed once with 100 μL of buffer A and redispersed in 100 μL of buffer A. Biotinlyated aptamers at 6.6 μM (100 μL) were heated at 80 oC for 3 min, cooled at room temperature, and then was added to the above suspension of beads. The mixture of the aptamers and magnetic beads incubated for 2 h under gently shaking. The magnetic beads were separated from the unreacted reagents by the magnetic separator, and the reaction solution was collected for the UV measurement of the concentration of the unreacted aptamer. The obtained aptamer modified magnetic beads were washed with buffer B containing 0.1% Tween 20 three times. The aptamer modified magnetic beads were finally redispersed in 100 μL of buffer B containing 0.1% Tween 20, and stored at 4 oC prior to use. By measuring the UV absorbance at 260 nm the concentration of the aptamer solution could be determined. The amount of the aptamers attached on the magnetic beads was estimated by measuring the decrease of aptamer concentration in the aptamer solution after the aptamer immobilization. The total amount of the aptamer attached on the magnetic beads (1 mg) in 100 μL of final solution was about 640 picomole.
Enzyme reaction toward the cleavage of peptide substrate
To optimize the enzyme reaction toward the hydrolysis of the fluorogenic substrate, the effect of pH and CaCl2 on the enzyme reaction was tested. 20 μL of enzyme reaction solution containing the fluorogenic substrate was mixed with 5 μL of APC (1 nM), and the mixture was incubated at 37 oC for 2 h. Then 100 μL of 2% acetic acid was added into the solution. The fluorescence intensity of the obtained solution emitted at 465 nm with an excitation at 350 nm was recorded with a fluorometer (HITACHI, F4500). The slits for excitation and emission were both set at 10 nm. One 100-μL quartz cuvette was used as the sample cell in the fluorescence measurement. Without further statement, the concentration of the fluorogenic substrate was 0.1 mM and the reaction solution contained 50 mM Tris-HCl (pH 8.0) and 150 mM NaCl.
Fig. S1 Effect of substrate concentration on enzyme reaction. Enzyme solution (20 μL) containing various concentrations of fluorogenic substrate was mixed with 5 μL of APC (1 nM), and the mixture was incubated at 37 oC for 2 h. The solution was transferred into 100 μL of 2% acetic acid solution, and the fluorescence at 465 nm with an excitation at 350 nm. The net increase RFU over the control that did not contain APC was plotted with the concentrations of the substrate.
The Michaelis constant Km was estimated by using Michaelis-Menten equation through the non-linear regression with the software of Graph Pad Prism. In the Michaelis-Menten equation, y=Vmaxx/(x+Km), y is the velocity of enzyme reaction, x is the concentration of substrate in the reaction solution, and Vmax is the maximum velocity. In the regression analysis, the obtained RFU change was used to represent the velocity. The obtained Km was 338±56 μM (R2=0.996). With 95% confidence interval, the Km was in the range from 194 μM to 482 μM.
Fig. S2 Aptamer-capture based assay for APC using aptamer modified magnetic beads and the fluorogenic substrate. 250 μL of APC samples at various concentrations were detected. (a) The fluorescence spectra obtained from the generated fluorescent products by APC. From the bottom to the top, the concentrations of APC were 0, 0.4, 1, 2, 4, 10, 20, 40, 100, 200, and 400 pM. The inset shows the detailed fluorescence spectra obtained from the generated fluorescent products by APC at 0, 0.4, 1, 2, 4, and 10 pM. (b) The relationship between the fluorescence change at 465 nm over the control and the concentration of APC
Fig. S3 Test of sequence specificity of the aptamer by using the control DNA modified magnetic beads. The control DNA modified magnetic beads were prepared by the same procedure for the aptamer modified magnetic beads. The same procedure for detection of APC (5 μL) with aptamer modified magnetic beads was applied for the analysis of APC at varying concentrations with the control DNA modified magnetic beads.
Fig. S4 Specificity test of the assay for APC using the fluorogenic substrate. 5 μL of the tested protein was added to 20 μL of reaction solution containing fluorogenic substrate (100 μM). After 2-h enzyme reaction at 37 oC, 100 μL of 2% acetic acid solution was added into the reaction solution, and the fluorescence intensity of the obtained solution emitted at 465 nm with an excitation at 350 nm was recorded. The concentrations of APC, thrombin, chymotrypsin, trypsin, elastase, and proteinase K were 1 nM.
Table S1. Comparison of various assays for APC with respect to methods, materials and reagents, limit of detection (LOD), linear dynamic range (LDR), and complex sample tested.
Enzyme assay / Chromogenic substrate / 100 pM / NA / not tested / 15-min enzyme reaction / 1
Enzyme assay / Fluorogenic substrate / 0.8 pM / 0.8 pM-100 pM / not tested / 20-min enzyme reaction / 2
Antibody-based enzyme assay / Antibody coated microplate, chromogenic substrate / 0.4 nM / 0.4 nM-90 nM / plasma / 1-h enzyme reaction / 3
Antibody-based enzyme assay / Antibody modified beads, chromogenic substrate / 40 pM
(200 μL) / 40 pM-1.8 nM / plasma / 4.5-h enzyme reaction / 4
Antibody-based enzyme assay / Antibody coated microplate, chromogenic substrate / 40 pM
(100 μL) / NA / plasma / 3-h enzyme reaction / 5
ELISA / Antibody coated microplate, PCI, peroxidase-labeled antibody for PCI / 2 pM / NA / plasma / 6
ELISA / Antibody coated microplate, PCI, antibody for PCI, streptavidin horse peroxidase conjugate / 3 pM
(200μL) / 3 pM-30 pM / plasma / mouse APC was detected / 7
Aptamer-based sandwich electrochemical assay / RNA aptamer on magnetic beads, RNA aptamer as reporter, streptavidin-alkaline phosphatase / 1 nM
(50 μL) / 10 nM-100 nM / not tested / 8
Aptamer-based assay on microplates / DNA aptamer on microplate, fluorogenic substrate / 0.39 pM
(100 μL) / NA / plasma / 4-h enzyme reaction / 9
Aptamer-based assay on magnetic beads / DNA aptamer on magnetic beads, fluorogenic substrate / 0.4 pM (250μL) / 0.4 pM-0.2 nM / diluted serum / 2-h enzyme reaction / this work
PCI, protein C inhibitor; NA, not available
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