Bioanalytical Chemistry

From Biomolecular Recognition to Nanobiosensing

Paolo Ugo, Pietro Marafini, Marta Meneghello

• Publisher: De Gruyter
• Year: 2021
• Print ISBN: 9783110589092
• eBook ISBN: 9783110589290
• Edition: 1
• Topic: Technology & Engineering
• Subtopic: Biochemistry
• Index: Technology & Engineering

Bioanalytical chemistry plays today a central role in various fields, from healthcare to food and environmental control. This book presents the main methodologies for analyzing biomacromolecules, with a focus on methods based on molecular recognition. The six chapters move from fundamentals to the most recent advances, achieved by a synergetic combination of bio and nanotechnologies. The need for rapid and reliable analytical tools able to perform a large number of quantitative analyses, not only in centralized laboratories and core facilities but also for point-of-care testing, has been dramatically stressed by the recent crisis caused by the COVID-19 pandemic. The aim of the authors is to provide graduate students and young researchers with the elements of interdisciplinary knowledge necessary not only to use the wide arsenal of bioanalytical tools available today but also to contribute to the development of even more effective devices and methods.

1 Biomacromolecules in analytical chemistry

1.1 Nucleic acids

The discovery of the substance that would later be known as deoxyribonucleic acid (DNA) was made by Johann Friedrich Miescher in 1869. He extracted the nuclei of leucocytes, taken from the pus on fresh surgical bandages, and he obtained a precipitate called “nuclein”. When the acidic properties of “nuclein” were discovered, the name was changed to “nucleic acid” by Richard Altmann in 1889. Most importantly, in 1944 Oswald, MacLeod and McCarty suggested that DNA carries genetic information, and their finding was confirmed in 1952 by Alfred Hershey and Martha Chase. A year later, James D. Watson and Francis H. C. Crick proposed the double helix structure for DNA.

1.1.1 Structure of nucleotides

DNA and RNA are biological polymers built up from monomers called nucleotides that are linked together by phosphodiester linkages. Nucleotides have three main components: a nitrogen-containing heterocyclic base, a pentose sugar (2′-deoxy-D-ribose in DNA and D-ribose in RNA) and a phosphate (Figure 1.1). There are five different fundamental nitrogenous bases and only four are present in DNA. These are divided into two groups (Figure 1.2):

• the purines: adenine (A) and guanine (G);
• the pyrimidines: cytosine (C), thymine (T) and uracil (U).

Figure 1.1: Structure of nucleotides. The pentose here depicted is 2′-deoxy-D-ribose (Adapted from P. Marafini, Biophysical Studies of Oligonucleotides Containing Duplex Stabilising Modifications, DPhil Thesis, University of Oxford, 2017. Reproduced with permission from the author).

Figure 1.2: The five nitrogenous bases present in DNA and RNA. Thymine is present in DNA only, while uracil is present only in RNA. The numbering of the nucleobases is in light blue (Adapted from P. Marafini, Biophysical Studies of Oligonucleotides Containing Duplex Stabilising Modifications, DPhil Thesis, University of Oxford, 2017. Reproduced with permission from the author).

The nucleobase is linked to the pentose sugar via an N-glycosidic bond connecting the C-1′ of the sugar with N-9 of purines or N-1 of pyrimidines. The nitrogenous base can be located on the same side of the pentose ring as its 5′-hydroxyl group (β-anomer) or on the opposite side (α-anomer), with naturally occurring oligonucleotides presenting nucleosides in the β-configuration (Figure 1.3).

Figure 1.3: α- and β-anomers of a DNA nucleoside (Adapted from P. Marafini, Biophysical Studies of Oligonucleotides Containing Duplex Stabilising Modifications, DPhil Thesis, University of Oxford, 2017. Reproduced with permission from the author).

The nucleobase can rotate around the N-glycosidic bond, having two extreme conformations (Figure 1.4). The syn conformer has the bigger N-3 (purine) or O-2 (pyrimidine) above the pentose ring, and it is therefore more sterically hindered than the anti conformer that has the smaller H-8 (purine) or H-6 (pyrimidine) above the sugar. For this reason, naturally occurring nucleic acids prefer the anti conformation.

Figure 1.4: Anti and syn conformations of deoxyadenosine (Adapted from P. Marafini, Biophysical Studies of Oligonucleotides Containing Duplex Stabilising Modifications, DPhil Thesis, University of Oxford, 2017. Reproduced with permission from the author).

In order to reduce electronic and steric interactions between the substituents of furanose, the ring is twisted out of plane generating the so-called sugar puckering. There are two forms of sugar pucker, identifiable by the position of the 2′ and 3′ carbons of the pentose ring (Figure 1.5). The South (S) configuration presents the C-2′ on the same side of the ring (endo) as the C-5′, while the North (N) configuration presents the C-3′ in the endo position. In DNA, 2′-deoxy-D-ribose usually adopts the C-2′ endo (S) conformation, while in RNA, D-ribose usually prefers the C-3′ endo (N) conformation.

Figure 1.5: C-2′ endo and C-3′ endo sugar conformations (Adapted from P. Marafini, Biophysical Studies of Oligonucleotides Containing Duplex Stabilising Modifications, DPhil Thesis, University of Oxford, 2017. Reproduced with permission from the author).

1.1.2 Structure of oligonucleotides and duplexes

One of the fundamental steps in understanding DNA structure was made by Erwin Chargaff who noted that the ratio of purines to pyrimidines in cells is equal (i.e. 1:1). This was followed by X-ray diffraction studies by Rosalind Franklin and Maurice Wilkins, which showed that the secondary structure of DNA is helical and humidity dependent. The key discovery in consolidating this information was published in 1953 by Jam...

Table of contents

• Title Page
• Copyright
• Contents
• Preface
• Abbreviation list
• Symbol list
• 1 Biomacromolecules in analytical chemistry
• 2 Introduction to bioanalytical assays and biosensors
• 3 Enzymatic biosensors
• 4 Immunochemical assays and immunosensors
• 5 Analysis of nucleic acids
• 6 Nanotechnologies for bioanalysis
• Index