POLYELECTROLYTE ENZYME-BEARING
MICRODIAGNOSTICUM––A NEW STEP
IN CLINICAL-BIOCHEMISTRY ANALYSIS
SERGEY A. TIKHONENKO, ALEXEI V. DUBROVSKY,
EKATERINA A. SABUROVA, and LYUDMILA I. SHABARCHINA
CHAPTER 8
CONTENTS
8.1 Introduction .....................................................................................................140
8.2 Experimental Details .......................................................................................140
8.2.1 Reagents ............................................................................................. 140
8.2.2 Stability of Encapsulated Enzymes Against Proteinase K .................141
8.3 Discussion and Results ...................................................................................141
8.3.1 Shell Formation with a Selected Pair of Polyelectrolytes ..................141
8.3.2 The Dependence of Proteins’ Distribution within Polyelectrolyte
Microcapsules on pH of the Medium .................................................143
8.3.3 Activity and Catalytic Characteristics of Encapsulated Enzymes .....144
8.3.4 Activity of Free and Encapsulated Enzymes during
Long-Term Storage and under the Action of Proteinase K ................ 146
Conclusions ..............................................................................................................147
References ................................................................................................................148
140 Engineering of Polymers and Chemical Complexity
8.1 INTRODUCTION
Clinical and biochemical analyzes are among the most common methods used to di-
agnose human diseases. Investigations of this kind are known to include general blood
and urine tests, the study of a number of other biological fluids [1-3]. Until recently,
these tests were carried out by chemical methods, but, due to the toxicity of many of
them, the low sensitivity and other shortcomings, enzymological methods widespread
received today. However, along with the obvious advantages of this approach, there
are some drawbacks: The ambiguity of the analysis in the presence of aggressive high-
molecular compounds to the enzyme, in particular, proteases and other intracellular
components; one-time use of the enzyme, and so on. Thus, there is a need to protect
the enzyme from the adverse effects, while maintaining access to it of the substrate, to
increase its stability during prolonged storage, as well as to develop the reusability of
the enzyme [4]. One type of such a defense is encapsulation of enzymes in polyelec-
trolyte microcapsules (PMC) [5,6].

this area is booming around the world: in the U.S., EU, China, Australia, and another
countries. This, along with pure fundamental research of structure, physico-chemical
and biological properties of polyelectrolyte microcapsules, increasing emphasis on
applied research aimed at practical use of PMC, particularly in medicine, chemical en-
gineering, biotechnology and many other areas [7]. The combination of unique proper-
ties and relatively simple technology of preparation for a wide range of PMC with the
given parameters (structural, mechanical, and functional), simplicity of inclusion of
a wide variety of substances, and the ability to control membrane permeability PMC
makes promising their use as tools for targeted drug delivery to the organs and tissues,
[8-13], depot [8] and the therapeutic effect, as microreactors, microcontainers.
The PMC containing enzyme can be used as a microdiagnosticum to detect a sub-

and in wasted water.
8.2 EXPERIMENTAL DETAILS
8.2.1 REAGENTS
Pig skeletal muscle lactate dehydrogenase (EC 1.1.1.27) (M4 isoform) was isolated
as in [14]. Urease (EC 3.5.1.5, jack bean) and proteinase K (EC 3.4.21.64) were from
Fluka (no. 94 285) and Sigma (P8044), respectively. The initial reagents used in the
preparation of the samples were as follows: Sodium poly(styrenesulfonate) (70 kDa),
poly(diallyldimethylammonium chloride) (70 kDa), and poly(allylamine hydrochlo-
ride) (70 kDa) from Aldrich (Germany); dextran sulfate (10 kDa) and EDTA from
Sigma (Germany); and calcium chloride, sodium carbonate, and sodium chloride from
Reakhim (Russia). The polyelectrolyte solutions were brought to a specified pH value
with concentrated solutions of NaOH or HCl.
The CaCO
3
microspherulites containing enzymes were prepared as in [6]. One vol-
ume of a 1 M CaCl
2
aqueous solution was added to two volumes of enzyme solution
(1.0 mg/ml) with vigorous stirring. Then an equal volume of aqueous 0.33 M Na
2
CO
3

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