Cellobiose dehydrogenase on electrodes - an electrochemical biosensor for various analytes tunable by positive charges

Abstract

Cellobiose dehydrogenase (CDH) is a sugar oxidizing enzyme secreted by various species of wood degrading fungi to assist the process of wood degradation. It can oxidise analytically relevant sugars as cellobiose, lactose or glucose leading to a gain of two electrons per sugar molecule. CDH consists of a flavin containing catalytic domain (DH) and a haem containing electron mediating domain (CYT). CDH is able to directly communicate with electrode surfaces via the CYT delivering its gained electrons making it a suitable candidate for the construction of mediatorless biosensors and biofuel cell anodes being applicable either for the detection of sugars or for the generation of electricity out of sugar containing solutions.

In the present thesis the ability of CDH to electrically communicate with silver, gold and graphite electrode surfaces was investigated and employed mainly by electrochemical techniques as cyclic voltammetry and square wave voltammetry and was complemented by spectroscopic techniques.

The central finding is the ability of cations to enhance the electro-catalytic activity of CDH. Especially divalent cations as Ca2+ were found to increase the internal electron transfer (IET) from the DH to the electron mediating CYT leading to higher current outputs. The effect was ascribed to a yet unknown, transient, electrostatic interaction of Ca2+ with negative charges present on the DH and CYT decreasing their repulsion leading to a faster IET. Similar effects were observed for CDH electrodes premodified with immobilised polycations as polyethylenimine (PEI) or polydiallyldimethylammonium chloride (PDADMAC) or premodified with PEI covered gold nanoparticles. The polycations were found to enhance the enzyme load onto electrode surfaces by electrostatic interactions but were also suggested to increase the IET comparable to Ca2+. The beneficial effect of cations and polycations on the electro-catalytic activity of CDH was employed to construct various biosensors to detect lactose, glucose, adenosine triphosphate and Ca2+ in various sensing schemes and analytes.

A further observation regarding the electrochemistry of CDH could be obtained only recently. We could finally prove unequivocally, after more than a decade of efforts, that a direct electronic communication is also possible between electrodes and the DH domain occurring at lower voltages than the DET with CYT. This potentially increases the voltage of biofuel cells and lowers the problematic oxidation of common interferents of biosensors pushing the commercial exploitation of CDH as a bioelectrocatalyst to a new level.

Abstract (Swedish)

Popular Abstract in English

Enzymes are nanometer sized work units made up of proteins. Each of the thousands of the existing enzymes are produced by the cells of every living organism. They carry out certain functions as putting molecules together, e. g. connect sugar units to form cellulose for a growing tree or destroy molecules, e. g. degrade cellulose into its sugar units to eat and live on them. The latter is done by wood degrading fungi. One enzyme produced by such fungi and involved in the degradation process of wood is cellobiose dehydrogenase (CDH) – the main actor in this thesis, which is depicted on the cover of this thesis. The function of CDH is to substract electrons from cellobiose sugar units and deliver them to another enzyme, which makes highly reactive radicals with the help of those electrons and oxygen. Those oxygen radicals help to degrade the very stable wood. In this thesis CDH was put on electrode surfaces made of graphite and gold. Instead of delivering the electrons to the radical producing protein the electrons were delivered now to the electrode instead and the amount of gained electrons was counted by measuring the electrical current, which is nothing else than a flow of electrons. This is called a biosensor. Little amounts of sugar only give little currents and vice versa. So measuring the current one can calculate how much sugar is there. This is useful if one for example wants to know how much lactose is there in milk or how much sugar is there in blood. This is exactly how diabetric patients measure their blood sugar nowadays, only with a less fancy enzyme. In this thesis we found out that we can enhance the speed of how fast CDH can transport the electrons within the protein to the electrode by simply adding calcium ions, which are positively charged. That gave us more electric current so that we could measure sugar much more accurately. Also other positively charged compounds as long chained polymers gluing CDH to the electrode surface or to gold nanoparticles did the same effect. Our research showed that this effect depends on from which wood degrading fungus the enzyme was taken from, on the acidity of the measuring solution and on the calcium ion or polymer concentration. This is good to know because later sensors are supposed to only measure sugar units and should not be disturbed by calcium ions.

In this thesis we also managed to open up another electron transfer pathway from deep inside the enzyme to the electrode. This helped us to get the electrons with less of an applied force, which is beneficial for the CDH biosensor because the less force we apply the smaller the chance of accidently substracting electrons from other molecules, which we don’t want to measure, like vitamin C.

We also used a CDH biosensor to measure the release of lactose from ibuprofen tablets. Most tablets contain lactose as a filling material, only little of the tablet is the actual drug. So knowing how fast the lactose is released helps to understand how fast the tablet dissolves in the body.

Another biosensor was made to measure ATP, the common energy currency in your body. But this was only possible using two other enzymes working together with CDH in an assembly line like manner on the electrode surface.

The developed sensors are only prototypes – one cannot buy them. But in the near future CDH based biosensors are planned to be really used for measuring glucose in blood or lactose in dairy factories. And I look forward to participating in this.

• author: Schulz, Christopher
• supervisor: Lo Gorton
• opponent: Lojou, Elisabeth, Centre national de la recherche scientifique (CNRS), Marseille, France
• organization: Biochemistry and Structural Biology
• publishing date: 2015
• type: Thesis
• publication status: published
• subject: Analytical Chemistry
• keywords: cellobiose dehydrogenase, direct electron transfer, amperometric biosensor, glucose, lactose, cellobiose, calcium, polycation, graphite, gold, electrode
• pages: 260 pages
• defense location: Kemicentrum, lecture Hall A, Getingevägen 60, Lund
• defense date: 2015-09-25 13:15:00
• ISBN: 978-91-7422-409-2
• language: English
• LU publication?: yes
• id: 504a0b47-757f-4c16-b851-658770e0fe67 (old id 7767510)
• date added to LUP: 2016-04-04 10:59:13
• date last changed: 2018-11-21 21:01:58