bioinspired design of a polymer gel sensor for the realization of extracellular ca 2+ imaging - polymer gel powder

by:Demi     2019-09-07
bioinspired design of a polymer gel sensor for the realization of extracellular ca 2+  imaging  -  polymer gel powder
Although the role of extra-cellular calcium as a messenger of inter-cellular communication is gaining increasing attention, there is currently no tool available for imaging calcium dynamics in the out-of-cell region.
Here we report the first solid
State-of-the-state fluorescent calcium sensor that meets the basic requirements for achieving out-of-cell calcium imaging.
Inspired by the Natural Calcium
Sensing receptors, we designed a special type of chemistry-
Cross-linked polyacrylic gel for monomer polymerization
Chain aggregation in the presence of Ca 2.
By additional aggregation-
The induced emission luminous agent is emitted to polyacrylic acid as a pendant, and the conformational state of the main chain is successfully converted into fluorescent properties at a given CA 2 concentration.
The calcium sensor has Nano-moles-
The ordered apparent dissociation constant compatible with the extracellular Ca 2 concentration, and showed sufficient dynamic range and excellent selectivity in the presence of physiological concentrations of biologically related ions, therefore, it is possible to monitor the sub-molar fluctuation of Ca 2 in the flow analytes containing the concentration of millions of mol ca 2.
Unless otherwise stated, all commercial reagents are used for the purposes received. Prior to use, -BA ()Acrylic ()
, Diacrylic acid of Sigan alcohol (), 1,4-
Alcohol diacrylic acid ()and 1,10-
Diacrylic acid diester ()
Purification by AlO column to remove polymerization inhibitors.
Azobisisobutyronitrile (AIBN)and ,′-methylenebis-acrylamide ()
Re-crystal and purification with methanol. 4-(1,2,2-triphenylvinyl)phenol ()
It was prepared according to the procedure of the report. (See ). A CHCl (5u2009mL)
Chlorine-propylene based solution (0. 42u2009mM, 5. 2u2009mmol)
Drop to CHCl at 0 °c (30u2009mL)
4-solution of mixture(1,2,2-triphenylvinyl)phenol (
910 mg, 35 mg)
Triamine (EtN, 1. 46u2009mL, 10u2009mmol).
Stir the resulting mixture at 25 °c for 3 Thanh, pour it into the saturated aqueous solution of NaHCO, and then extract it with CHCl.
Wash the organic extract with water and salt water, dry on the waterless MgSO, and then evaporate to dry under decompression.
Column chromatography with residue (
SiO, hexane/CHCl 1/1 v/v)
Allow isolation of as white solids (756u2009mg)
Yield at 72%: h nmr (400u2009MHz, CDCl)(ppm)7. 01–7. 11 (m, 15H), 6. 89 (d, u2009=u20099. 0u2009Hz, 2H), 6. 56 (dd, u2009=u200917. 3, 1. 3u2009Hz, 1H), 6. 27 (dd, u2009=u200910. 5, 17. 3u2009Hz, 1H), 5. 99 (dd, u2009=u200910. 5, 1. 3u2009Hz, 1H). C NMR (100u2009MHz, CDCl)(ppm)164. 3, 149. 0, 143. 7, 143. 6, 143. 5, 141. 4, 141. 3, 140. 0, 132. 4, 132. 3, 131. 4, 131. 3, 128. 1, 127. 9, 127. 8, 127. 7, 126. 6, 126. 5, 120. 7. FT-IR (KBr)ν (cm)
3076,3054, 3024,1756, 1677,1599, 1502,1443, 1356,1200, 1166,1140, 1017,763,748,699,613,572,498. HRMS (FAB): calcd. for CHO [M]/u2009=u2009402. 1620; found: /u2009=u2009402. 1617.
Usually,DMF)solution (1. 53u2009mL)
Single mixture (21. 3u2009mg, 53u2009mol), -BA (, 146u2009L, 1. 0u2009mmol)and AIBN (1. 7u2009mg, 11u2009mol)
It's frozen. pump-thaw cycles (three times)
Clean with nitrogen.
The mixture is stirred at 60 °c for 12 °c, cooled to 25 °c, and then evaporated to dry under decompression.
The residue was frozen.
Dry from tolueneBu-PAA-
Quantitative TPE white solid (167u2009mg): H NMR (400u2009MHz, CDCl)(ppm)6. 79–7. 11 (br), 2. 05–2. 39 (br), 1. 71–1. 86 (br), 1. 20–1. 63 (br). FT-IR (KBr)ν (cm)
2979,2935, 1731,1481, 1457,1393, 1368,1257, 1149,1034, 909,846,751,701,471,430.
Use a program similar to the followingBu-PAA-TPE, -Bu-PAA-
Quantitative acquisition of TPE from monomer ,-BA ()
And AIBN of the corresponding monomer feed ratio.
-Values and PDIBu-PAA-
The TPE evaluated by predictive analysis is summarized as follows.
Normally, trifluoride acetic acid (58u2009L)was added to -Bu-PAA-TPE (10. 0u2009mg, 78u2009mol).
The mixture is stirred for 12 hours at 25 °c and evaporated to dry under decompression.
The residual volatile compounds are removed by methanol boiling device (
100 mL, five times)to afford PAA-
Quantitative white solid (9. 8u2009mg): H NMR (400u2009MHz, CDOD)(ppm)6. 79–7. 21 (br), 2. 28–2. 65 (br), 1. 40–2. 22 (br). FT-IR (KBr)ν (cm)
2961, 2361, 1716, 1503, 1454, 1417, 1249, 1168, 802, 701, 614, 503, 414.
Use a program similar to PAA-TPE, PAA-
Quantification of TPE from trifluoride acetic acid and corresponding precursor (-Bu-PAA-TPE).
Component ratio of PAA-
The TPE evaluated by the hydrogen nuclear magnetic resonance spectrum is summarized as follows.
Usually,0. 71u2009mL)
Solution of a single mixture (21. 3u2009mg, 53u2009mol)Acrylic (, 67 µL, 79mol)
, Diacrylic acid of Sigan alcohol (, 8. 6u2009L, 32u2009mol)and AIBN (1. 7u2009mg, 11u2009mol)
It's frozen. pump-thaw cycles (three times)
Clean with nitrogen.
The mixture is allowed to place 12 °c at 60 °c and then cool to 25 °c.
The obtained gel-like materials were extracted by Sauth with methanol/acetone mixture (1/1u2009v/v)
24 hours, dry at 80 °c for 48 hours at a reduced pressure, providing g-PAA-
White solids of thermoplastic elastomer (46u2009mg)in 48% yield.
The program used is similar to g-PAA-TPE, g-PAA-
About 50% of yield is obtained from single acrylic acid ()
, Diacrylic acid of Sigan alcohol ()
And AIBN of the corresponding monomer feed ratio.
Preparation of the feed ratio of g-PAA-
Overview of thermoplastic elastomer.
Use similar to g-PAA-
Other cross-linked polymers (TPE))
Yield of about 50% from single acrylic acid ()
The corresponding Chain Home (–)
And AIBN of the corresponding monomer feed ratio.
Ca titration curve was obtained by measuring fluorescence intensity (for PAA-TPE)
Or quantum yield (for g-PAA-TPE)
At various Ca concentrations.
Because the number of valid Ca-
Binding sites in PAA-TPE and g-PAA-
Disassociation cannot be determined and is used to determine the general metrological analysis of the dissociation constant ()
Not applicable to these systems.
Instead, we used the apparent, which was obtained by fitting the Ca titration curve with the Hill equation below (1)
Using Least squares in R software ().
: Fluorescence intensity (for PAA-TPE)
Or quantum yield (for g-PAA-TPE)
: Maximum fluorescence intensity (for PAA-TPE)
Or quantum yield (for g-PAA-TPE)
: Minimum fluorescence intensity (for PAA-TPE)
Or quantum yield (for g-PAA-TPE)
: Apparent dissociation constant: apparent Hill coefficient of gel expansion, slice sample immersed in HEPES buffer solution (70u2009mM, pHu2009=u20097. 4)
30 minutes at 25 °c.
The swelling rate is evaluated according to the following equation (2)
: Weight of dry g-PAA-
Swollen: weight of Swollen g-PAA-
According to NIH, the TPEAll experimental program was approved by the Animal Care Committee of Nara Medical University (USA)
Guidelines and guidelines for the proper conduct of animal experiments published by the Japanese scientific committee.
The details of the experiment are described.
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