220
T. Zhang et al.
Chain 1:5’-TCGGCACGTTCTCAGTAGCGCTCGCTGGTCATCCCACAGC
TACGT-3’
Chain 2:5’-GGACGCGGTTAGGGGAGAATTTCCATACCTTTGGGTTGTA3’
8.3.2.1 Aptamer Assays in Vitro
HIV ribonuclease H (RNase H) is a key marker of HIV infection. RNase H is an
endonuclease which adjusts gene regulation in living cells by hydrolyzing RNA
in DNA/RNA chimeric strands [65]. Traditional method for determining RNase H
activity is polyacrylamide gel electrophoresis (PAGE), which is laborious and timeconsuming, however. Recently, the development of RNA aptamer, RNA mimics of
green fluorescent protein (GFP) has revolutionized RNase research in fluorescencerelated studies. RNA aptamers of 3, 5-difluoro-4-hydroxybenzylidene imidazolinone
(DFHBI) that can specially bind to the GFP-like target fluorophores have generated
a new fluorescence assay in RNase detection. Spinach is one of the stable aptamers
binding to DFHBI. Based on it, Xiegroup [63] pioneered to employ the GFP-like
RNA-fluorophore complexes to build a fluorescence biosensor for the activity assay
of HIV RNase H (Fig. 8.5a). A very low LOD was achieved at 0.0018 U/mL with
the linear range of 0.001–100 U/mL. The assay utilizing GFP-like structure to bind
to DFHBI developed a new fluorescent detection scheme and formed an excellent
modality for the label-free labeling.
Colloidal gold labeling immunochromatographic test strip is a mature and applicable technology at present. Wang et al. [66] developed quantum dots-based fluorescent lateral flow assay strips to detect HIV-DNA in human serum. This assay realized
a detection limit as slow as 0.76 pM and detection range of 1 pM to 10 nM. It is
obvious that the designed fluorescent testing strips can act as a detection measure
for early diagnosis and management of AIDS. Besides fluorescent-based detection,
colorimetric assay has also attracted considerable attention for infections diagnosis in
qualitative and quantitative analyses because of its low cost and simplicity diagnosis
[64, 67, 68]. Fatin et al. [64] explored an unmodified gold nanoparticle (GNP)-based
colorimetric approach by using the non-functional split aptameroligos (Fig. 8.5b). In
the assay, the color transitions from red to purple in relation to the dose-dependency
of HIV-1. The visible color transition was characterized using UV–vis spectrophotometer and scanning electron microscopy. The limit of detection of HIV-1 Tat was
as low as 10 nM.
8.3.2.2 Probes for Imaging of HIV RNase Activity in Living Cells
Spinach, RNA aptamer of DFHBI that binds to the GFP-like target fluorophores,
has also generated a new kind of RNA-fluorophore complexes for cellular imaging
research. Xie group [63] constructed RNA mimics green fluorescent protein strategy
for probing RNase H activity in single cell. In vitro, HIV RNase activity can be
T. Zhang et al.
Chain 1:5’-TCGGCACGTTCTCAGTAGCGCTCGCTGGTCATCCCACAGC
TACGT-3’
Chain 2:5’-GGACGCGGTTAGGGGAGAATTTCCATACCTTTGGGTTGTA3’
8.3.2.1 Aptamer Assays in Vitro
HIV ribonuclease H (RNase H) is a key marker of HIV infection. RNase H is an
endonuclease which adjusts gene regulation in living cells by hydrolyzing RNA
in DNA/RNA chimeric strands [65]. Traditional method for determining RNase H
activity is polyacrylamide gel electrophoresis (PAGE), which is laborious and timeconsuming, however. Recently, the development of RNA aptamer, RNA mimics of
green fluorescent protein (GFP) has revolutionized RNase research in fluorescencerelated studies. RNA aptamers of 3, 5-difluoro-4-hydroxybenzylidene imidazolinone
(DFHBI) that can specially bind to the GFP-like target fluorophores have generated
a new fluorescence assay in RNase detection. Spinach is one of the stable aptamers
binding to DFHBI. Based on it, Xiegroup [63] pioneered to employ the GFP-like
RNA-fluorophore complexes to build a fluorescence biosensor for the activity assay
of HIV RNase H (Fig. 8.5a). A very low LOD was achieved at 0.0018 U/mL with
the linear range of 0.001–100 U/mL. The assay utilizing GFP-like structure to bind
to DFHBI developed a new fluorescent detection scheme and formed an excellent
modality for the label-free labeling.
Colloidal gold labeling immunochromatographic test strip is a mature and applicable technology at present. Wang et al. [66] developed quantum dots-based fluorescent lateral flow assay strips to detect HIV-DNA in human serum. This assay realized
a detection limit as slow as 0.76 pM and detection range of 1 pM to 10 nM. It is
obvious that the designed fluorescent testing strips can act as a detection measure
for early diagnosis and management of AIDS. Besides fluorescent-based detection,
colorimetric assay has also attracted considerable attention for infections diagnosis in
qualitative and quantitative analyses because of its low cost and simplicity diagnosis
[64, 67, 68]. Fatin et al. [64] explored an unmodified gold nanoparticle (GNP)-based
colorimetric approach by using the non-functional split aptameroligos (Fig. 8.5b). In
the assay, the color transitions from red to purple in relation to the dose-dependency
of HIV-1. The visible color transition was characterized using UV–vis spectrophotometer and scanning electron microscopy. The limit of detection of HIV-1 Tat was
as low as 10 nM.
8.3.2.2 Probes for Imaging of HIV RNase Activity in Living Cells
Spinach, RNA aptamer of DFHBI that binds to the GFP-like target fluorophores,
has also generated a new kind of RNA-fluorophore complexes for cellular imaging
research. Xie group [63] constructed RNA mimics green fluorescent protein strategy
for probing RNase H activity in single cell. In vitro, HIV RNase activity can be