1 3
Topics in Current Chemistry (2020) 378:13
high specificity and affinity (about 10
3
- to 10
6
-fold higher than an antigen–antibody
interaction [103]). Biotin-based conjugates are also easy to synthesize and have less
impact on the activity of the biomolecules. However, despite its enormous advantages and wide applicability, avidin has several limitations, including non-specific
binding and possible immunogenicity. To avoid these limitations, concentrated
efforts have been devoted to discovering and engineering superior variants of avidin.
Information on the structure and physical–chemical properties of such variants are
described in detail by de Freitas et al. [25, 104].
The specific binding of MNPs to DNA using high-affinity biotin–streptavidin
noncovalent interactions requires preliminary modification of the surface of MNPs
and nucleic acids with the appropriate molecules [17]. For example, Cannon et al.
designed streptavidin-modified IONPs to bind biotinylated ssDNA-labeled with
a Cy3 fluorescent dye [105] and thus enhance the immobilization process. The
authors concluded that such nanocomposites are sufficiently specific to allow amplification-free detection of DNA and RNA molecules in drawn blood samples. Similarly, He et al. reported the use of chemiluminescence detection of alkaline phosphatase–streptavidin capable of binding superparamagnetic IONPs modified with
biotin-labeled hepatitis B virus DNA [106]. In this particular case, the functionalization of MNPs with (3-aminopropyl) triethoxysilane (APTES) allowed further treatment with succinic anhydride to efficiently bind the biotinylated DNA.
4 Biomedical and Technological Applications of MNPs
4.1 Magnetic Separation of DNA
Separation technology is one of the most complex and important areas of biotechnology research [107]. It is considered to be the starting point for downstream processes and product development and comprises molecular routine biotechnological
activities such as DNA sequencing, amplification, cloning, and bio-detection [108].
Generally, successful nucleic acid purification requires four important steps: (1)
effective disruption of cells or tissue; (2) denaturation of nucleoprotein complexes;
(3) inactivation of nucleases (e.g., DNase for DNA extraction); and (4) secure storage away from any possibility of contamination.
Traditional extraction and DNA purification techniques can be divided into two
fundamental categories, namely, those in which purification is mediated with organic
solvents and those in which purification occurs by solid-phase methodologies.
Among those techniques in which purification is mediated with organic solvents,
the guanidinium thiocyanate–phenol–chloroform extraction technique is considered
to be a conventional method that comprises the formation of a biphasic emulsion
to purify DNA. In short, two layers are formed by centrifugation, with one layer, a
mixture of phenol–chloroform, used to internalize proteins, carbohydrates, and cell
debris, and the second layer, the aqueous phase, containing the purified DNA molecules. The DNA is then precipitated using ethanol or isopropanol in 2:1 or 1:1 ratio
and a high salts concentration [107]. Similar techniques, such as alkaline extraction
29
Reprinted from the journal
Topics in Current Chemistry (2020) 378:13
high specificity and affinity (about 10
3
- to 10
6
-fold higher than an antigen–antibody
interaction [103]). Biotin-based conjugates are also easy to synthesize and have less
impact on the activity of the biomolecules. However, despite its enormous advantages and wide applicability, avidin has several limitations, including non-specific
binding and possible immunogenicity. To avoid these limitations, concentrated
efforts have been devoted to discovering and engineering superior variants of avidin.
Information on the structure and physical–chemical properties of such variants are
described in detail by de Freitas et al. [25, 104].
The specific binding of MNPs to DNA using high-affinity biotin–streptavidin
noncovalent interactions requires preliminary modification of the surface of MNPs
and nucleic acids with the appropriate molecules [17]. For example, Cannon et al.
designed streptavidin-modified IONPs to bind biotinylated ssDNA-labeled with
a Cy3 fluorescent dye [105] and thus enhance the immobilization process. The
authors concluded that such nanocomposites are sufficiently specific to allow amplification-free detection of DNA and RNA molecules in drawn blood samples. Similarly, He et al. reported the use of chemiluminescence detection of alkaline phosphatase–streptavidin capable of binding superparamagnetic IONPs modified with
biotin-labeled hepatitis B virus DNA [106]. In this particular case, the functionalization of MNPs with (3-aminopropyl) triethoxysilane (APTES) allowed further treatment with succinic anhydride to efficiently bind the biotinylated DNA.
4 Biomedical and Technological Applications of MNPs
4.1 Magnetic Separation of DNA
Separation technology is one of the most complex and important areas of biotechnology research [107]. It is considered to be the starting point for downstream processes and product development and comprises molecular routine biotechnological
activities such as DNA sequencing, amplification, cloning, and bio-detection [108].
Generally, successful nucleic acid purification requires four important steps: (1)
effective disruption of cells or tissue; (2) denaturation of nucleoprotein complexes;
(3) inactivation of nucleases (e.g., DNase for DNA extraction); and (4) secure storage away from any possibility of contamination.
Traditional extraction and DNA purification techniques can be divided into two
fundamental categories, namely, those in which purification is mediated with organic
solvents and those in which purification occurs by solid-phase methodologies.
Among those techniques in which purification is mediated with organic solvents,
the guanidinium thiocyanate–phenol–chloroform extraction technique is considered
to be a conventional method that comprises the formation of a biphasic emulsion
to purify DNA. In short, two layers are formed by centrifugation, with one layer, a
mixture of phenol–chloroform, used to internalize proteins, carbohydrates, and cell
debris, and the second layer, the aqueous phase, containing the purified DNA molecules. The DNA is then precipitated using ethanol or isopropanol in 2:1 or 1:1 ratio
and a high salts concentration [107]. Similar techniques, such as alkaline extraction
29
Reprinted from the journal