[269–271]. The early in vitro studies on PON1, where it was shown that it was able
to hydrolyze paraoxon, were on the basis that led to the adoption of the name [271–
273]. Thorough studies on the hydrolysis of over 50 substrates have concluded that
the native activity of PON1 was that of a lactonase, with promiscuous phosphotriesterase and paraoxonase activities [270, 274]. Regarding its use in the construction of biosensing devices, while having in mind the wide variety of substrates
that are reported to be metabolized by this hydrolase and their analytical interest, its
application as a biorecognition element is rather non-existent in the literature. In
fact, only two devices have been reported recently featuring the enzyme in such a
role [275, 276]. Both analytical tools were targeted towards paraoxon and diazon
detection, using either an optical or electrochemical transduction method,
respectively.
As an alternative to OPH, lipases (triacylglycerol ester hydrolases) can be
employed as the catalytic biorecognition element. These enzymes catalyze the
hydrolysis and synthesis of long-chain acylglycerols and many other esters [277,
278]. Unlike OPH, lipases are commercially available at a more affordable price,
making them an attractive alternative [277]. Lipases can also be used as an alternative to cholinesterases (ChE), which are the most common hydrolases used in
inhibition-based organophosphate determination [268]. This was demonstrated in
the amperometric determination of chlorfenvinphos and malathion using the
decrease in lipase’s activity towards p-nitrophenyl acetate as a function of the
pesticides’ concentration [279]. ChE-based devices are designed using a mono-, biand tri-enzymatic detection system. In the first, artificial substrates such as
acetylthiocholine or butyrylthiocholine are used, and the hydrolysis product thiocholine is detected via oxidation [280, 281]. In the bi-enzymatic system, ChE is
coupled to the flavoenzyme ChOx. The hydrolysis product choline is oxidized by
ChOx and detection is made either through O 2 depletion or H 2 O 2 oxidation monitoring [101, 282]. Lastly, the tri-enzymatic approach adds peroxidase to the previous, and detection is made by the catalytic reduction of H 2 O 2 [283].
Apart from organophosphorus compounds monitoring, hydrolase-based
amperometric/voltammetric biosensors have also been developed for clinical
diagnostic application and metals detection. For example, urease which catalyzes
the hydrolysis of urea to ammonia has been used as the key element in
amperometric/voltammetric devices for urea [284–288], lead [289], and mercury
[289–291] determination. The enzyme is commonly immobilized in a conducting
polymer matrix (polypyrrole or polyaniline) that serves a dual purpose. First, it
provides a good microenvironment for enzyme immobilization; secondly, its
interaction with the produced ammonia makes it possible to detect the urea
hydrolysis reaction using amperometric/voltammetric methods [285, 287, 292].
Another example of hydrolase-based clinical tools is that of multienzyme biodevices for triglycerides determination, in which lipase is the key biorecognition
element. Together with glycerol kinase and glycerol-3-phosphate oxidase, lipase
catalyzes the hydrolysis of triglycerides to fatty acids and glycerol, followed by
oxidation of the later, producing H 2 O 2 and locally consuming O 2 [293–296].
Alternatively, lipase can be combined with glycerol dehydrogenase, which oxidizes
Selective Enzymes at the Core of Advanced Electroanalytical …
343
to hydrolyze paraoxon, were on the basis that led to the adoption of the name [271–
273]. Thorough studies on the hydrolysis of over 50 substrates have concluded that
the native activity of PON1 was that of a lactonase, with promiscuous phosphotriesterase and paraoxonase activities [270, 274]. Regarding its use in the construction of biosensing devices, while having in mind the wide variety of substrates
that are reported to be metabolized by this hydrolase and their analytical interest, its
application as a biorecognition element is rather non-existent in the literature. In
fact, only two devices have been reported recently featuring the enzyme in such a
role [275, 276]. Both analytical tools were targeted towards paraoxon and diazon
detection, using either an optical or electrochemical transduction method,
respectively.
As an alternative to OPH, lipases (triacylglycerol ester hydrolases) can be
employed as the catalytic biorecognition element. These enzymes catalyze the
hydrolysis and synthesis of long-chain acylglycerols and many other esters [277,
278]. Unlike OPH, lipases are commercially available at a more affordable price,
making them an attractive alternative [277]. Lipases can also be used as an alternative to cholinesterases (ChE), which are the most common hydrolases used in
inhibition-based organophosphate determination [268]. This was demonstrated in
the amperometric determination of chlorfenvinphos and malathion using the
decrease in lipase’s activity towards p-nitrophenyl acetate as a function of the
pesticides’ concentration [279]. ChE-based devices are designed using a mono-, biand tri-enzymatic detection system. In the first, artificial substrates such as
acetylthiocholine or butyrylthiocholine are used, and the hydrolysis product thiocholine is detected via oxidation [280, 281]. In the bi-enzymatic system, ChE is
coupled to the flavoenzyme ChOx. The hydrolysis product choline is oxidized by
ChOx and detection is made either through O 2 depletion or H 2 O 2 oxidation monitoring [101, 282]. Lastly, the tri-enzymatic approach adds peroxidase to the previous, and detection is made by the catalytic reduction of H 2 O 2 [283].
Apart from organophosphorus compounds monitoring, hydrolase-based
amperometric/voltammetric biosensors have also been developed for clinical
diagnostic application and metals detection. For example, urease which catalyzes
the hydrolysis of urea to ammonia has been used as the key element in
amperometric/voltammetric devices for urea [284–288], lead [289], and mercury
[289–291] determination. The enzyme is commonly immobilized in a conducting
polymer matrix (polypyrrole or polyaniline) that serves a dual purpose. First, it
provides a good microenvironment for enzyme immobilization; secondly, its
interaction with the produced ammonia makes it possible to detect the urea
hydrolysis reaction using amperometric/voltammetric methods [285, 287, 292].
Another example of hydrolase-based clinical tools is that of multienzyme biodevices for triglycerides determination, in which lipase is the key biorecognition
element. Together with glycerol kinase and glycerol-3-phosphate oxidase, lipase
catalyzes the hydrolysis of triglycerides to fatty acids and glycerol, followed by
oxidation of the later, producing H 2 O 2 and locally consuming O 2 [293–296].
Alternatively, lipase can be combined with glycerol dehydrogenase, which oxidizes
Selective Enzymes at the Core of Advanced Electroanalytical …
343