decrease (“switch off”). By adding guest, dye could be displaced out of the host cavity,
which reverses fluorescent “switch on” or “switch off.” Based on this indicator
displacement principle, fluorescent analytical method based on host-guest chemistry
was developed. Two host-dye complexes were used for the assay: CB[7]-Dapoxyl
complex as a “switch-on” indicator and p-sulfonatocalix[4]arene-DBO complex as a
“switch off” indicator. These two supramolecular complexes were used to monitor
the enzymatic process of amino acid decarboxylation catalyzed by decarboxylase.
Catalytic product is a stronger guest for macrocyclic host and could displace
encapsulated dye, which results in change in fluorescence intensity. This method
was successfully applied to continuously track decarboxylase enzymatic activity.
Robustness of the assay was tested based on CX4 sensor system in the presence of
different buffers and common additives as well as its dependence on the dye/host
ratio by varying the dye concentration. It was discovered that in all cases, three- to
eightfold increase in fluorescence was observed, indicating that the enzyme was
catalytically active. This new assay concept is complementary to other assays
involving membrane channels and calorimetric microarrays and offers a promising
alternative to the broad use of individually raised and subsequently labeled antibodies. Considering the vast amount of water-soluble as well as organic-soluble
synthetic receptors with different affinities, this assay principle could also be applied
to other systems and sensing purposes.
Fluorescent dye-based indicator displacement assay was pushed forward to cell
study. As shown in Fig. 4, Norouzy et al. used p-sulfonatocalix[4]arene and N,
N
0 -dimethyl-9,9
0 -biacridinium dinitrate (lucigenin, LCG) as a “switch off” reporter
pair [14]. Biological and pharmaceutical molecules acetylcholine, choline, and
protamine were used as analytes. Reporter pair and analyte were sequentially
uptaken by live cells. By imaging cells with fluorescence microscopy, it was
discovered that fluorescence intensity enhanced significantly when analyte was
uptaken. It was proposed that encapsulated dye was displaced by analyte. This
work demonstrated that indicator displacement assay could be a powerful tool to
detect suitable analyte in live cells. To monitor concentration and distribution of
biological or medicinal molecules is important and yet challenging. The supramolecular method reported in this work indicated guest displacement might offer new
route to achieve this goal in a noninvasive and continuous manner.
23.2.4 Pillar[n]arenes
PAs are a new type of macrocyclic host molecules with pillar-shaped architecture
[9]. PAs are generally prepared by one-pot condensation reaction of pmethoxybenzene and paraformaldehyde with methylene bridges on para-positions.
PAs of variable sizes have been developed with PA[5]-PA[7] as the most frequently
used homologues.
PAs are hydrophobic macrocyclic host molecules. By introducing carboxylic acid
groups or amino groups to PAs, water-soluble PAs or WPs have been developed with
high solubility in water. WPs could encapsulate small-molecule guests by multiple
23 Stimuli-Responsive Self-Assembly Based on Macrocyclic Hosts and Biomedical. . .
609
which reverses fluorescent “switch on” or “switch off.” Based on this indicator
displacement principle, fluorescent analytical method based on host-guest chemistry
was developed. Two host-dye complexes were used for the assay: CB[7]-Dapoxyl
complex as a “switch-on” indicator and p-sulfonatocalix[4]arene-DBO complex as a
“switch off” indicator. These two supramolecular complexes were used to monitor
the enzymatic process of amino acid decarboxylation catalyzed by decarboxylase.
Catalytic product is a stronger guest for macrocyclic host and could displace
encapsulated dye, which results in change in fluorescence intensity. This method
was successfully applied to continuously track decarboxylase enzymatic activity.
Robustness of the assay was tested based on CX4 sensor system in the presence of
different buffers and common additives as well as its dependence on the dye/host
ratio by varying the dye concentration. It was discovered that in all cases, three- to
eightfold increase in fluorescence was observed, indicating that the enzyme was
catalytically active. This new assay concept is complementary to other assays
involving membrane channels and calorimetric microarrays and offers a promising
alternative to the broad use of individually raised and subsequently labeled antibodies. Considering the vast amount of water-soluble as well as organic-soluble
synthetic receptors with different affinities, this assay principle could also be applied
to other systems and sensing purposes.
Fluorescent dye-based indicator displacement assay was pushed forward to cell
study. As shown in Fig. 4, Norouzy et al. used p-sulfonatocalix[4]arene and N,
N
0 -dimethyl-9,9
0 -biacridinium dinitrate (lucigenin, LCG) as a “switch off” reporter
pair [14]. Biological and pharmaceutical molecules acetylcholine, choline, and
protamine were used as analytes. Reporter pair and analyte were sequentially
uptaken by live cells. By imaging cells with fluorescence microscopy, it was
discovered that fluorescence intensity enhanced significantly when analyte was
uptaken. It was proposed that encapsulated dye was displaced by analyte. This
work demonstrated that indicator displacement assay could be a powerful tool to
detect suitable analyte in live cells. To monitor concentration and distribution of
biological or medicinal molecules is important and yet challenging. The supramolecular method reported in this work indicated guest displacement might offer new
route to achieve this goal in a noninvasive and continuous manner.
23.2.4 Pillar[n]arenes
PAs are a new type of macrocyclic host molecules with pillar-shaped architecture
[9]. PAs are generally prepared by one-pot condensation reaction of pmethoxybenzene and paraformaldehyde with methylene bridges on para-positions.
PAs of variable sizes have been developed with PA[5]-PA[7] as the most frequently
used homologues.
PAs are hydrophobic macrocyclic host molecules. By introducing carboxylic acid
groups or amino groups to PAs, water-soluble PAs or WPs have been developed with
high solubility in water. WPs could encapsulate small-molecule guests by multiple
23 Stimuli-Responsive Self-Assembly Based on Macrocyclic Hosts and Biomedical. . .
609