Minjung Kim

Last Homework

Bioengineering Biosensors
1) [50%] A protein-functionalized sensor for the capture and detection of a specific protein,
hapten, immunoglobulin, viral or bacterial analyte.
Acute myocardial infarction (AMI) is one of the leading causes of death in the United States, and its
prevalence is increasing worldwide. Currently, there are three main biomarkers for the disease: myoglobin
(MG), cyto-kinase MB (CK-MB), and cTnT. When the three biomarkers are present simultaneously, it is
a good indicator that the patient most likely has AMI. There is a need for an assay that detects the three
biomarkers in one test to save time and money. A stripping voltammetric immunoassay that uses a
Quantum Dot (QD) label has been done recently with the use of magnetic beads to detect the presence
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of three distinct cancer biomarkers . I propose that the same method be done to detect the three main
biomarkers of AMI. (Note: please refer to Appendix A for the full names of the acronyms used in the
figures)

DynaBeadss epoxy rings can be crosslinked to the amines of on the Fc region of monoclonal antibodies.
It is a common procedure outlined in the product manual.

Minjung Kim
Last Homework
PAMAM dendrimers (D) can act as a label that attaches to the second antibody. Dendrimers with a
sulfhydryl group can be washed with metal ions and sulfide ions to create three distinctive dendrimer QD
1,2

labels.

Once the label is made, polyclonal antibodies can be crosslinked with heterofunctional sulfo-

SMCC (active groups are sulfo NHS ester and maleimid). The amine located in the Fc region will react
with the sulfo NHS ester functional group. Then the maleimide will crosslink with the sulfhydryl group of
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the dendrimer QD label.

When monoclonal antibodies are crosslinked with the labeled biomarkers, a stripping voltammetric
immunoassay can be carried out by washing the solution with HNO3 to precipitate out the metal ions.
Then after magnetic separation, a three electrode with Ag/AgCl reference electrode can be used to make
square wave anodic stripping voltammetric (SWASV) measurements where current was measured as
increasing voltages were applied. In the presence of the biomarkers, three distinctive peaks will be
observed at three different voltages: peaks for MG, cTnT, and CK-MB will be found at -1.2V, -0.75V, and 0.45V respectively. More importantly, the magnitude of the peak will be proportional to the concentration
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of the biomarkers present.

Minjung Kim
Last Homework
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Set up of the Anodic Stripping Voltammetry (ASV) :

3) [50%] A DNA-functionalized sensor for the capture of a nucleic acid or DNA-binding protein
Hepatitis virus C (HVC) is a viral disease that cause Hepatitis C, a chronic liver disease. The
disease can also put patients in high risk for liver cancer. Over half a million people die from Hepatitis C
annually, and currently there are no vaccines available. As a result, early detection of the disease is best
to treat the patient. A DNA functionalized piezoelectric Quartz Crystal Microbalance (QCM) biosensor is a
label free sensor that can detect Hepatitis C by the characteristic DNA sequence.

The characterized DNA sequence associated with HVC can be used as the probe DNA. It can react with
biocytin hydrazide to so that the biotin is attached on the 5 prime end of the single strand DNA
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sequence.

Minjung Kim
Last Homework

The crystal will be plated with gold electrodes. A monolayer of cysteamine can form on the surface of the
gold electrode plated quartz. Then glutaraldehyde can be added to activate the amine group. Then a
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Schiff base can form between Avidin and aldehyde. The Schiff base can be stabilized using sodium
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borohydride. When the DNA

conjugated with biotin is added to the
gold electrodes, the Avidin will non
covalently interact with biotin.
Although the specific interaction is
not known, it is very specific and
strong enough because biotin-Avidin
interaction is known to be the
strongest non covalent bond
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observed. Like most solids, QCM

has a specific resonant frequency.
This resonant frequency changes as
mass on the crystal change. In this
assay, the change in resonant
frequency is measured which is an
indirect result of target DNA binding
to the probe DNA and thus changing
the mass on the quartz crystal.

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Setup for the assay :

Minjung Kim
Last Homework
References
1. Tang D, Hou L, Niessner R, Xu M, Gao Z, Knopp D. Multiplexed electrochemical immunoassay of
biomarkers using metal sulfide quantum dot nanolabels and trifunctionalized magnetic beads.
Biosensors and Bioelectronics. 2013 Aug 15; 46:37-43.
2. Crooks RM, Zhao M, Sun L, et al. Dendrimer-encapsulated metal nanoparticles:synthesis,
characterization, and application to catalysis. Accounts of Chemical Research. 2001 Mar;
34(3):181-90.
3. Wangler C, Moldenhauer G, Eisenhut M, et al. Antibody-dendrimer conjugates: the number, not
the size of the dendrimers determines the immunoreactivity. Bioconjugate Chemistry. 2008 Apr;
19(4): 813-20.
4. Kokkinos C, Economou A, Petrou PS, et al. Microfabricated Tin-Film Electrodes for Protein and
DNA Sensing Based on Stripping VOltammetric Detection of Cd(II) Released from Quantum Dots
Labels. Analytical Chemistry. 2013 Oct; 85(22):10686-91.
5. Skladal P, dos Santos Riccardi, et al. Piezoelectric biosensors for real-time monitoring of
hybridization and detection of hepatitis virus C. Journal of virological methods. 2004 May;
117(2):145-51.
6. Reisfeld A, Rothenberg JM, et al. Nonradioactive hybridization probes prepared by the reaction
of biotin hydrazide with DNA. Biochemical and biophysical research communications. 1987 Jan
30; 142(2):519-26.
7. Xiao Y, Ju HX, Chen HY. Hydrogen peroxide sensor based on horseradish peroxidase-labeled Au
colloids immobilized on gold electrode surface by cysteamine monolayer. Analytical Chemistry.
1999 May;26(1):72-82.
8. Billman JH, Diesing AC. Reduction of Schiff Bases with Sodium Borohydride. The Journal of
Organic Chemistry. 1957 22(9):1068-1070.
9. Gitlin G, Khait I, Bayer EA, et al. Studies on the biotin-binding sites of avidin and streptavidin. A
chemically induced dynamic nuclear polarization investigation of the status of tyrosine residues.
Biochemical Journal. 1989 Apr;259(2):493-498.
10. Yao C, Zhu T, Tang J, et al. Hybridization assay of hepatitis B virus by QCM peptide nucleic acid
biosensor. Biosensors and Bioelectronics. 2008 Jan 18;23(6):879-85.
Appendix A
Full name

Acronym

Magnetic Epoxy Bead

MB

human Myoglobin

MG

Creatine Kinase MB

mAb CK-MB

Dendrimer

Monoclonal antibody

mAb

Polyclonal antibody

Ab

Metal ion

Metal sulfide

MS