New trends in ion-selective electrodes - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 88

The protamine ion-selective electrode used as an end-point detector created another
possibility for the determination of heparin concentration via titration with protamine,
utilizing the specifi c heparin-protamine interaction. Ramamurthy et al. [42] reported
on an improved protamine-selective electrode. The polymeric membrane was doped
with the ion exchanger dinonylnaphthalenesulfonate (DNNS), which demonstrated bet-
ter performance in comparison to previously used tetrakis-(4-chlorophenyl)borate [38].
The electrode exhibited signifi cant non-equilibrium potentiometric response to pro-
tamine over the concentration range of 0.5-20 mg l 1 in undiluted whole blood samples.
Whole blood heparin concentrations determined by titration ( n 157) correlated well

with other methods, including ACT (the Hepcon HMS) ( r 0.934) and a previously

reported potentiometric heparin sensor-based method [41] ( r 0.973). Reasonable cor-

relation was found with a commercial chromogenic anti-Xa heparin assay ( r 0.891)

with corresponding plasma samples and appropriate correction for hematocrit levels.
Titration with protamine using protamine-selective electrode as an end-point detector
was also shown to be a reliable methodology for the detection of low-molecular-weight
heparins (LMWH) in whole blood samples at concentrations up to 2 U mL 1 [43]
(measured heparin activity is in units per milliliter according to the US Pharmacopeia
standard). These results were especially important because commercially avail-
able LMWH samples are very heterogeneous, since they are prepared using different
methods such as chemical and enzymatic hydrolysis of unfractionated heparin (UFH).
Results of determination of LMWH biological activity in whole blood are normally not
reliable because ACT assays are not always sensitive toward LMWHs [44]. The anti-Xa
assay, the most adopted methodology for detection of LMWH, can be performed only
with plasma samples and thus cannot be used as a point-of-care method.
Among other polyions Meyerhoff's group demonstrated successful detection of
pentosan polysulfate (PPS) [45], an anti-osteoarthritis drug, in plasma and polyanions
with high phosphate content, including polyphosphates and nucleic acids in saline
solutions [46].
Due to their response mechanism the polyion-selective electrodes are not sensi-
tive to the small fragments of polyionic macromolecules. Thus, if an enzyme cleaves
the polyionic molecule these sensors can be used for detection of enzyme activity.
Polycation protamine is rich in arginine residues that make it a suitable substrate for
protease-sensitive electrochemical assays. Real-time detection of trypsine activity was
demonstrated with the protamine-selective electrode as a detector [38].
Protamine is also known as a specifi c substrate for plasmin, the proteolytic enzyme
produced by the trombolytic cascade [47]. It was demonstrated that the protamine-
selective electrode can be used as a sensor for the detection of thrombolytic agents such
as urokinase in human plasma [48]. Urokinase or plasminogen alone being added to
the protamine solution did not produce a change in electrode potential. However, a sig-
nifi cant decrease in the potential was observed when both urokinase and plasminogen
were added to the protamine solution, suggesting that the conversion of plasminogen to
plasmin by urokinase occurred followed by the digestion of protamine by plasmin.
This same approach was used for assaying other protease activities such as chy-
motrypsin and renin [49]. Chymotrypsin and renin cleave only at specifi c sites of