Specific binding force measurement using microfluidic system
Date Issued
2015
Date
2015
Author(s)
Chen, Chi-Yu
Abstract
We measured the specific binding force between proteins with the background of the development of heart disease detection biochips. In clinical, it is pathogonostic to have acute coronary syndrome (ACS) of three items that are clinical symptoms, electrocardiography (ECG) diagnosis and cardiac enzymes. To achieve the goal of constructing a point-of-care system for ACS patients, this thesis is dedicated to the development of biological sensing chip. Specifically, aptamers capture the heart blood enzyme (Cardiac troponin I) in order to detect the concentration of the enzyme contained in blood. Since the biochip was combined with the microfluidic channel, the blood was delivered into the sensing area through pressure difference by vacuum or the capillary force. The shear force generated by fluid could break the binding between proteins, so we are committed to measuring the binding force between specific bindings and developing a system with high accuracy to investigate different kinds of protein-protein interactions. The method of measuring the interaction force between proteins had been revealed by many literatures. However, most of them used atomic force microscope (AFM) to measure the force. This method of measurement need well controlled environment. Alternatively, we developed a microfluidic system to measure the binding force between primary antibody and secondary antibody indirectly. In our research, the primary antibody (Rabbit IgG) was covalently bonded to carboxylated (-COOH) microbeads. The substrate of the microchannel was deposited with a film of polymer containing NHS-ester functional group by chemical vapor deposition, and the secondary antibodies (Anti-rabbit IgG) can be covalently bonded on the substrate through these functional groups. The microbeads conjugated with the primary antibodies were injected into the microchannel and captured by the secondary antibodies on the substrate. Then the buffer was injected into the microchannel with gradually increased flow rate. When the shear force was large enough to break the bonds, the microbeads will be washed away. In addition, we quantified the numbers of proteins on one bead with the use of fluorescent labeled protein by fluorescence intensity measurement. With the above experiment and model calculations, the binding force between the primary antibody and the secondary antibody can be calculated. In the measurement results, the binding force between the antibodies was 118 pN. Though the result is greater than the binding force between human IgG and anti-human IgG measured from other literatures, it was still very close. The measured interaction force is attributed to the bond, van der Waals force and electrostatic force. If the van der Walls force and electrostatic force can be eliminated by calculation, the force only contributed by the bond can be investigated.
Subjects
binding force measurement
aptamer
cardiac troponin I
acute coronary syndrome
rabbit IgG
anti-rabbit IgG
microfluidic
shear force
microbead
SDGs
Type
thesis
File(s)![Thumbnail Image]()
Loading...
Name
ntu-104-R02522506-1.pdf
Size
23.54 KB
Format
Adobe PDF
Checksum
(MD5):ce1fb4244175a1028318ef2689432b03