Biosensors: Electrochemical Devices-General Concepts and Performance

Abstract

This review provides a general overview of different biosensors, mostly concentrating on electrochemical analytical devices, while briefly explaining general approaches to various kinds of biosensors, their construction and performance. A discussion on how all required components of biosensors are brought together to perform analytical work is offered. Different signal-transducing mechanisms are discussed, particularly addressing the immobilization of biomolecular components in the vicinity of a transducer interface and their functional integration with electronic devices. The review is mostly addressing general concepts of the biosensing processes rather than specific modern achievements in the area.

Keywords: electrochemical biosensors; electron-transfer mediators; enzyme immobilization; enzyme-based biosensors; signal transducers.

Figure 1

Schematic presentation of a biosensor. The analyte (bio)molecules can be selectively analyzed in a complex mixture containing many other molecules (different molecules are shown schematically with different shapes). The specificity in the analyte detection is provided by biomolecular species immobilized at the signal transducer interface. The electronic component of the biosensor provides the conversion of a chemical signal to an electronic signal with its amplification. The larger size of the second arrow shows an amplified signal. The computer, used in modern devices, allows convenient presentation of the output signal and its processing as needed for specific applications. Notably, the computer is usually miniaturized and integrated with the electronic signal processing part, not being a desktop device, as shown in the scheme for simplicity.

Figure 2

Transduction methods for converting analyte molecule signals to an electronic output: (i) optical measurements based on absorbance or fluorescence spectroscopy, (ii) various electrochemical measurements including amperometry, potentiometry, impedance spectroscopy, etc., (iii) semiconductor measurements, e.g., field-effect transistor signal transduction, using current, potential or capacitance measurements, (iv) surface plasmon resonance (SPR) measurements, (v) microgravimetric measurements using quartz crystal microbalance (QCM), and (vi) nanotechnological methods, e.g., atomic force microscope (AFM) measurements.

Figure 3

Enzyme-catalyzed reactions can be used for the transformation of a substrate-analyte signal into an electronic signal. The scheme shows an example of such enzyme-based signal transduction using cholesterol oxidase for the analysis of cholesterol. A side H₂O₂ product is detected electrochemically, e.g., by amperometric technique. While the measurements are based on the analysis of H₂O₂, its concentration is stoichiometrically related to the cholesterol concentration in the analyzed sample. Notably, another approach can be based on an analysis of the O₂ depletion instead of the H₂O₂ production.

Figure 4

The biomolecule analyte signal biocatalytically converted to pH change, then analyzed with a pH meter operating as a signal transducer. The scheme shows the conversion of acetylcholine analyte to choline and acetic acid catalyzed by acetylcholinesterase enzyme. Biocatalytic signal processing is an example of a general biosensor approach based on pH changes related to the analyte concentration (note that the process should be performed without a buffer solution to allow the pH change). Numerous biosensors based on the same approach have been constructed using the coupling of biocatalytic reactions with measured pH changes. The conventional pH-sensitive glass electrode is shown for simplicity. In real biosensors, miniaturized pH sensors are used. In practical applications, the acetylcholinesterase enzyme inhibitors are analyzed instead of the substrate analysis.

Figure 5

The scheme illustrates a “sandwich”-type immunoassay applied for the analysis of an antigen analyte. The primary antibody operating as a biorecognition species is immobilized at the transducer interface. When an antigen analyte appears in a solution, it produces an affinity antibody–antigen complex at the interface. The changes in the interface properties might be insufficient for their analysis, particularly when the antigen is represented by a small molecule. The secondary antibody labeled with a reporter unit reacts with the affinity complex, then is attached to the antigen analyte. The produced “sandwich” complex composed of antibody–antigen–antibody brings to the surface a reporter unit which produces an amplified output signal. Note that the complex is produced and the reporter unit is bound to the surface only when the antigen analyte is present. Otherwise, the secondary antibody is washed out and the signal from the reporter unit is not produced at the surface. The reporter unit (label) linked to the secondary antibody can be represented by an enzyme (e.g., horseradish peroxidase, HRP), a fluorescent dye, a quantum dot, a Au nanoparticle (NP), or a carbon nanotube (CNT). Some other labels (e.g., magnetic nanoparticles) are not shown in the scheme. The labels can generate an amplified signal in the form of color changes, fluorescence, electric response, microgravimetric response, etc. Respectively, the transducer can be optically transparent, electrically conducted, QCM, etc., depending on the signal generated by the label.

Figure 6

Kinetics of biomolecular recognition processes proceeding in biosensors: (a) in a homogeneous solution or (b) at a transducer interface. Reaction kinetics remains the same at t = 0 and t > 0 in the case of a homogeneous reaction; reaction rate constant = k₁ at t = 0 and t > 0. Reaction rate constant changes according to surface coverage in the case of a heterogeneous process because of the interactions of the neighboring species; reaction constant = k₂ at t = 0 and k₃ at t > 0.

Figure 7

Migration of biomolecule adsorbate with attractive or repulsive interactions.

Figure 8

The sequence of events at a surface leading to chemisorption.

Figure 9

Bifunctional reagents: (a–d) homobifunctional—containing two identical reacting groups for binding to the same functional groups of biomolecules; (e–g) heterobifunctional—containing two different reacting groups for binding to different functional groups of biomolecules. The following reacting groups are shown in the examples given: (a) aldehyde groups reacting with amino groups, (b,e,f,g) active ester groups reacting with amino or hydroxyl groups, (c,e) maleimide and (g) iodoacetyl groups reacting with thiol groups, and (d,f) azido groups used for click-chemistry reactions. The spacers separating the reacting groups can be flexible (a–d) or rigid (e–g), thus resulting in different attachment modes of biomolecules. The spacer length can be different, short or long, and some spacers can be cleavable due to S–S bonds (d) or other chemically or photochemically cleavable units.

Figure 10

A homobifunctional cross-linker containing two active ester functions used to bind biomolecules by reacting with their amino groups and producing amido bonds. Note the long and flexible spacer between the reacting groups. Many other similar cross-linkers are available with various structures and length of the spacer connecting the reacting groups.

Figure 11

A heterobifunctional cross-linker containing a maleimide function for binding to thiol groups of biomolecules and an active ester function for binding to amino groups of biomolecules. Note the short and rigid spacer between the reacting groups. Many other similar cross-linkers are available with various structures and lengths of the spacer connecting the reacting groups.

Figure 12

(A) Immobilization of a bioactive molecule onto a silane-derivatized substrate. (B) Immobilization of a silane-derivatized bioactive molecule.

Figure 13

(A) Schematically shown silanization of an indium tin oxide (ITO) electrode used in electrochemical biosensors. Note that glass surfaces can be modified similarly, while being used in optical biosensors. (B) The ITO electrode deposited onto a glass slide as a thin conducting film (note a rigid support for the ITO electrode). (C) The ITO electrode deposited onto a polymer film as a thin conducting film (note a flexible support for the ITO electrode).

Figure 14

(A) Multilayer coverage of an ITO electrode upon silanization with a silane with three hydrolyzable groups performed in protonic solvents (e.g., ethanol) or in aprotic solvents (e.g., toluene) with traces of water. (B) Strictly monolayer coverage of an ITO electrode using silane with one hydrolyzable group. (C) Deactivation of the terminal amino group upon formation of a cyclic structure in the silane layer. (D) Preserving an active terminal amino group in the silane layer with two amino groups in the silane molecule.

Figure 15

Self-assembly of thiol/disulfide molecules on a Au electrode, then introducing various functional groups (amino-, carboxyl- or active ester for covalent immobilization of (bio)molecules).

Figure 16

Kinetic steps of oxidase enzyme catalysis.

Figure 17

Examples of the oxidative and reductive soluble mediators shuttling electrons between electrodes and oxidative or reductive enzymes. Mediators can be used as “charge transfer messengers” between electrodes and enzymes.

Figure 18

Example of mediated reductive bioelectrocatalysis.

Figure 19

Bioelectrocatalytic processes involving different redox enzymes: (a) flavin adenine dinucleotide (FAD)-oxidases (exemplified with GOx), (b) NAD⁺/NADH-dehydrogenases (exemplified with NAD⁺-glucose dehydrogenase, NAD⁺-GDH); note that a catalyst, Cat, is usually required for recycling the NAD⁺/NADH cofactor), and (c) PQQ-glucose dehydrogenase (PQQ-GDH). All three enzyme classes are exemplified here with the enzymes oxidizing glucose.

Figure 20

NAD⁺/NADH cofactor structure in the oxidized (a) and reduced (b) states. Note that this cofactor is used by NAD⁺/NADH-dependent dehydrogenases.

Figure 21

Bioelectrocatalytic glucose oxidation by the system immobilized at an electrode surface. The system includes a polymeric matrix bound to the electrode surface and composed of a cross-linked polymer network with pendant redox mediator groups. The GOx enzyme is physically entrapped into the polymer matrix. The redox mediator used in the system is [Os(2,2′-bipyridine)₂]⁺ covalently bound to the (poly)4-vinylpyridine network.

Figure 22

(A) Reconstitution of GOx-apo-enzyme (enzyme with the removed active center) on a monolayer composed of an artificial analog of the FAD active centers. PQQ located between the active centers and the electrode surface operates as an intermediate electron-transfer “station” facilitating the electron transport to the electrode surface. (B) The cyclic voltammograms show the background current in the absence of glucose (a) and in the presence of glucose, 80 mM (b), potential scan rate, 5 mV s⁻¹. Abbreviations used: FAD—flavin adenine dinucleotide, PQQ—pyrroloquinoline quinone, EDC—1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (reagent used in the covalent immobilization process).

Figure 23

Amperometric glucose analysis with the use of NAD⁺-glucose dehydrogenase (GDH) and PQQ catalyst for recycling NADH back to NAD⁺. Note that this enzyme system is O₂-insensitive, thus being different from systems based on O₂-dependent glucose oxidase (GOx). Different catalysts, mostly represented by quinones, can be used to facilitate the electron transfer between NAD⁺/NADH-dependent enzymes and electrodes. The PQQ catalyst shown here is only an example.

Figure 24

Schematic of photosynthetic electron transport chain (PET) consisting of photosystem II (PSII) oxidizing H₂O and producing O₂, and photosystem I (PSI) reducing NADP⁺ to NADPH. P680 and P700 are special pairs composed of chlorophyll molecules being excited with light (P680* and P700* correspond to their excited states). Electron-transfer chain components are abbreviated. The inhibition center and mediated effect can be localized approximately in the middle of the electron-transfer chain between PSII and PSI.

Figure 25

Examples of the pH decrease (A) and increase (B) produced by enzyme reactions. Notably, in practical applications, the enzymes should be immobilized onto a pH-sensitive transducer surface.