1. Research Plan
Detection of Protamine and Heparin using Silver Nanoprisms
Elizabeth Isaac
Objective Statement
The detection of protamine and heparin can be done using silver nanoprisms as an
indicator in the solution. In this project, a silver nanoprism size and protamine and heparin
concentration based study will be done to understand the affinity of silver nanoprisms for
protamine and heparin. With further work, silver nanoprisms can be used in a detection system
that will enable safer use of heparin and protamine in medical treatments.
Intellectual Merit
While a lot of has been done with gold nanoparticles and their interaction with heparin
and protamine [1], there is not a lot of information about the interaction with silver. This project
will develop fundamental knowledge on how protamine and heparin interact with silver, a
cheaper and more stable alternative to gold.
Broader Impact
This project will allow undergraduates the opportunity to get hands on research
experience early on in their college career. The undergraduates will be able to learn how to
synthesis silver nanoprisms and analytical techniques such as UV-Vis spectroscopy.
Background/State of the Art
Heparin, a widely used injectable anticoagulant, is used to prevent the formation of clots
and is used in the treatment of a variety of medical conditions. Heparin has the highest known
charge density of any biological molecule and is massive with a molecular mass of 12,000-
15,000 g/mol [2]. While heparin is useful, it also has its fair share of issues. One serious side
effect of heparin use is heparin-induced thrombocytopenia[3]. This condition causes a decrease
in the amount of platelets in blood which causes fatigue, increased external bleeding, and a
decreased ability for the blood to form clots. As levels of heparin are difficult to monitor, it is
easy to cause an accidental heparin overdose in patients. These overdoses most commonly affect
children and are often fatal.
Protamine, a nucleic acid typically isolated from fish sperm, is used to counteract the
effects of a heparin overdose. It has its fair share of problems associated with its use as well,
including hypotension and anaphylactoid-like (allergic) reactions when administered too quickly
[4]. It is for these reasons that levels of protamine in a person’s system must be closely
monitored when used during treatments.
Using gold nanoparticles is the current state of the art for the detection of protamine and
heparin levels in blood. A safer alternative to gold is silver, which is not only cheap, but also has

2. Figure 1 [8]
several antibacterial, antiviral and, antitumor applications. Silver is also used in many biosensors
and optical-imaging applications [5]. It has been observed that due to several surface
enhancement factors, nanoprisms show better surface activity as compared to their spherical
counterparts. Figure 1. shows an image of size dependent silver nanoprism solution and a TEM
image of nanoprisms. Thus in this project we will study the size dependent affinity of silver
nanoprisms for different concentration levels of heparin and protamine. The affinity will be
measured by observing the change in the absorbance peak of silver nanoprism with the addition
of protamine and heparin
Hypothesis
1. Gold nanoparticles are currently used for the detection of
protamine and heparin. We hypothesize that due to the antiviral
and antibacterial properties, silver will be a better alternative for
protamine and heparin detection.
2. Protamine and heparin detection has always been done with
nanoparticles. We hypothesize that using silver nanoprisms will
decrease the limit of detection of the solution due to the surface
enhancement factors that has been observed in nanoprisms [6].
Methodology
Materials Required:
Sodium citrate (CAS no.18996-35-5), Silver nitrate (CAS no.7761-88-8), 30% Hydrogen
peroxide (CAS no.7722-84-1), Potassium bromide (CAS no.7758-02-3), Sodium borohydride
(CAS no.16940-66-2), Heparin (CAS no.9041-08-1) and Protamine (CAS no.9007-31-2) will be
purchased from Sigma Aldrich.
Nanoprism Synthesis: Silver nanoprisms will synthesized using the procedure described by
Frank et al [7]. To four vials the following was added in order: 2.0 ml of 1.25 x 10-2 M sodium
citrate, 5.0 ml of 3.75 x 10-4 M silver nitrate, and 5.0 ml of 5.0 x 10-2 M 30% hydrogen peroxide.
Then, to each vial 0 μl, 20 μL, 25 μl and 40 μl solution of 10-3 M potassium bromide will be
added respectively. A 5.0 x 10-3 M solution of sodium borohydride (2.5 ml) will be added to each
vial. The vials will be capped and stirred for 10 minutes for complete mixing. A change in color
of the solution will be monitored with respect to the volume of the potassium bromide added in
each vial. The reagents should all be mixed freshly right before synthesis as they are not stable
enough to be stored for future use.
Nanoprism Activity Measurement
The nanoprism activity for protamine and heparin will be measured using a UV-Vis
spectrometer. The absorbance and full width half maxima of the nanoprism will be measured
with and without the addition of heparin. Heparin will be added in several batches until there is

3. no change in the absorbance levels with the addition of heparin is observed. To the same cuvette,
protamine will be added and the change in absorbance will be measured. Also add protamine in
several batches until no change in absorbance is observed. The same study will be done on the
nanoprisms of all sizes
Conclusion
The addition of high negative charge density heparin in the positively charged silver
nanoprism solution will cause aggregation of the nanoprism solution. This aggregation can be
observed by a change in the UV-Vis absorption peak. Similarly, addition of positively charged
protamine into this solution will cause nanoprisms to disaggregate which will result in bringing
the absorption peak back to its original position. Plotting a calibration curve of heparin and
protamine concentration vs the size of nanoprisms will be great addition of knowledge in this
field. This is the first time a silver nanoprism size based study will be done for the detection of
protamine and heparin in a solution.
References:
1. Peng, X., et al., “Turn on-off” fluorescent sensor for protamine and heparin based on
label-free silicon quantum dots coupled with gold nanoparticles. Sensors and Actuators
B: Chemical, 2015. 213: p. 131-138.
2. Wardrop, D. and D. Keeling, The story of the discovery of heparin and warfarin. British
Journal of Haematology, 2008. 141(6): p. 757-763.
3. Arepally, G.M. and T.L. Ortel, Heparin-Induced Thrombocytopenia. New England
Journal of Medicine, 2006. 355(8): p. 809-817.
4. Shapira, N., et al., Cardiovascular effects of protamine sulfate in man. The Journal of
thoracic and cardiovascular surgery, 1982. 84(4): p. 505-514.
5. Huang, Z.H., et al., Controllable Synthesis and Biomedical Applications of Silver
Nanomaterials. Journal of Nanoscience and Nanotechnology, 2011. 11(11): p. 9395-
9408.
6. Ciou, S.-H., et al., SERS Enhancement Factors Studies of Silver Nanoprism and
Spherical Nanoparticle Colloids in The Presence of Bromide Ions. The Journal of
Physical Chemistry C, 2009. 113(22): p. 9520-9525.
7. Frank, A.J., et al., Synthesis of Silver Nanoprisms with Variable Size and Investigation of
Their Optical Properties: A First-Year Undergraduate Experiment Exploring Plasmonic
Nanoparticles. Journal of Chemical Education, 2010. 87(10): p. 1098-1101.
8. Millstone, J.E., et al., Colloidal Gold and Silver Triangular Nanoprisms. Small, 2009.
5(6): p. 646-664.