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On/off-switchable LSPR nano-immunoassay for troponin-T

View Article: PubMed Central - PubMed

ABSTRACT

Regeneration of immunosensors is a longstanding challenge. We have developed a re-usable troponin-T (TnT) immunoassay based on localised surface plasmon resonance (LSPR) at gold nanorods (GNR). Thermosensitive poly(N-isopropylacrylamide) (PNIPAAM) was functionalised with anti-TnT to control the affinity interaction with TnT. The LSPR was extremely sensitive to the dielectric constant of the surrounding medium as modulated by antigen binding after 20 min incubation at 37 °C. Computational modelling incorporating molecular docking, molecular dynamics and free energy calculations was used to elucidate the interactions between the various subsystems namely, IgG-antibody (c.f., anti-TnT), PNIPAAM and/or TnT. This study demonstrates a remarkable temperature dependent immuno-interaction due to changes in the PNIPAAM secondary structures, i.e., globular and coil, at above or below the lower critical solution temperature (LCST). A series of concentrations of TnT were measured by correlating the λLSPR shift with relative changes in extinction intensity at the distinct plasmonic maximum (i.e., 832 nm). The magnitude of the red shift in λLSPR was nearly linear with increasing concentration of TnT, over the range 7.6 × 10−15 to 9.1 × 10−4 g/mL. The LSPR based nano-immunoassay could be simply regenerated by switching the polymer conformation and creating a gradient of microenvironments between the two states with a modest change in temperature.

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(a) Extinction intensity change of GNR-anti-TnT (opened square) and GNR-anti-TnT-PNIPAAM (filled square) vs. anti-logarithm of TnT concentration, (b) UV-NIR spectra of GNR-anti-TnT-PNIPAAM LSPR nano-immunoassay when 1 (blue) and 15 (red) ng/mL TnT solutions were treated at 37 °C, rectangular (c′) zone is expanded to produce (c) left and right boundaries of green zones are the λLSPR for 1 and 15 ng/mL TnT solution respectively and (d) λLSPR shift vs TnT concentration in the range of from 1 to 15 ng/mL at 37 °C.
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f3: (a) Extinction intensity change of GNR-anti-TnT (opened square) and GNR-anti-TnT-PNIPAAM (filled square) vs. anti-logarithm of TnT concentration, (b) UV-NIR spectra of GNR-anti-TnT-PNIPAAM LSPR nano-immunoassay when 1 (blue) and 15 (red) ng/mL TnT solutions were treated at 37 °C, rectangular (c′) zone is expanded to produce (c) left and right boundaries of green zones are the λLSPR for 1 and 15 ng/mL TnT solution respectively and (d) λLSPR shift vs TnT concentration in the range of from 1 to 15 ng/mL at 37 °C.

Mentions: In the present investigation, UV-NIR studies suggest that the intensity of the longitudinal LSPR mode is very sensitive with respect to TnT concentration and can be used as a tool for monitoring the troponin release. A change in extinction intensity versus anti-logarithm of TnT concentration is shown in Fig. 3(a) for GNR-anti-TnT and GNR-anti-TnT-PNIPAAM conjugates. Extinction intensity differences before and after an addition of TnT were observed with a gradual increase in TnT concentration. The black line with filled squares and red line with opened squares correspond to extinction intensity changes for GNR-anti-TnT-PNIPAAM and GNR-anti-TnT conjugates, respectively. Both the traces depict an almost linear decrease in λLSPR intensity change with increasing of TnT concentration from 7.6 × 10−15 g/mL to 9.1 × 10−4 g/mL40. The change in extinction intensity is observed due to the weakening of the charges/ionic potential over the GNR surface by binding of antigen. At higher temperature (37 °C), the PNIPAAM configuration is changed from coil to globular shape which may offer sufficient space for association of TnT on to the surface of GNRs, as a results the extinction peak remains sharp, i.e., the shape before association of TnT except broadening. It is also consistent with an optical polarisation of ionic impulse and temperature responsive behaviour of the PNIPAAM lids at anti-TnT with respect to concentration of TnT. The anti-TnT optical immunoassay with optical polarisation of the ionic impulse was enhanced at the LCST of PNIPAAM in the GNR-anti-TnT-PNIPAAM for TnT sensing at biological temperature, i.e., 37 °C. It is interesting to note that the extinction intensity change in the case of GNR-anti-TnT-PNIPAAM is higher that the GNR-anti-TnT11. The enhanced extinction intensity was due to a change in the effective refractive index of the medium, which is mainly caused by the PNIPAAM of GNR-anti-TnT-PNIPAAM conjugates. A sharp change (rapid fall) in the extinction at a concentration, ln[TnT] equals to around ~15 is very likely attributed to that fact that incident light frequency nearly matches to the collective plasmon oscillations, as determined by the effective dielectric constant of the medium. As mentioned earlier, anti-TnT-PNIPAAM conjugates showed a maximum extinction (longitudinal) at a wavelength (λLSPR) ~831 nm. The peak in the longitudinal band broadened with increase of TnT concentration, while the transverse band only showed a decrease in extinction intensity with λLSPR shift of 1–2 nm at higher concentrations of TnT. The NIR spectra in the presence of two different representative TnT concentrations (1 and 15 ng/mL) are shown in Fig. 3(b), where λLSPR are observed at ~832 and ~854 nm respectively. The label-free LSPR based nano-immunoassay had a detection limit of 8.4 fg/mL with a response time of 10 sec. at 25 °C. The amplified rectangular area of Fig. 3(b) highlighted as 2(c′) is shown in Fig. 3(c) and the λLSPR for 1 and 15 ng/mL spectra is indicated by the left and right boundary of the green area, respectively. According to the results shown in Fig. 3(d), indicate the comparable values of peak shifting obtained with respect to the mentioned concentrations, which support the dissolution effects of TnT in a biological concentration. A red-shift in the λLSPR was noticed with the increase of the TnT concentration35. It is believed that higher TnT concentration changes effective material index eventually leading to a red shift in excitation peak.


On/off-switchable LSPR nano-immunoassay for troponin-T
(a) Extinction intensity change of GNR-anti-TnT (opened square) and GNR-anti-TnT-PNIPAAM (filled square) vs. anti-logarithm of TnT concentration, (b) UV-NIR spectra of GNR-anti-TnT-PNIPAAM LSPR nano-immunoassay when 1 (blue) and 15 (red) ng/mL TnT solutions were treated at 37 °C, rectangular (c′) zone is expanded to produce (c) left and right boundaries of green zones are the λLSPR for 1 and 15 ng/mL TnT solution respectively and (d) λLSPR shift vs TnT concentration in the range of from 1 to 15 ng/mL at 37 °C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5382532&req=5

f3: (a) Extinction intensity change of GNR-anti-TnT (opened square) and GNR-anti-TnT-PNIPAAM (filled square) vs. anti-logarithm of TnT concentration, (b) UV-NIR spectra of GNR-anti-TnT-PNIPAAM LSPR nano-immunoassay when 1 (blue) and 15 (red) ng/mL TnT solutions were treated at 37 °C, rectangular (c′) zone is expanded to produce (c) left and right boundaries of green zones are the λLSPR for 1 and 15 ng/mL TnT solution respectively and (d) λLSPR shift vs TnT concentration in the range of from 1 to 15 ng/mL at 37 °C.
Mentions: In the present investigation, UV-NIR studies suggest that the intensity of the longitudinal LSPR mode is very sensitive with respect to TnT concentration and can be used as a tool for monitoring the troponin release. A change in extinction intensity versus anti-logarithm of TnT concentration is shown in Fig. 3(a) for GNR-anti-TnT and GNR-anti-TnT-PNIPAAM conjugates. Extinction intensity differences before and after an addition of TnT were observed with a gradual increase in TnT concentration. The black line with filled squares and red line with opened squares correspond to extinction intensity changes for GNR-anti-TnT-PNIPAAM and GNR-anti-TnT conjugates, respectively. Both the traces depict an almost linear decrease in λLSPR intensity change with increasing of TnT concentration from 7.6 × 10−15 g/mL to 9.1 × 10−4 g/mL40. The change in extinction intensity is observed due to the weakening of the charges/ionic potential over the GNR surface by binding of antigen. At higher temperature (37 °C), the PNIPAAM configuration is changed from coil to globular shape which may offer sufficient space for association of TnT on to the surface of GNRs, as a results the extinction peak remains sharp, i.e., the shape before association of TnT except broadening. It is also consistent with an optical polarisation of ionic impulse and temperature responsive behaviour of the PNIPAAM lids at anti-TnT with respect to concentration of TnT. The anti-TnT optical immunoassay with optical polarisation of the ionic impulse was enhanced at the LCST of PNIPAAM in the GNR-anti-TnT-PNIPAAM for TnT sensing at biological temperature, i.e., 37 °C. It is interesting to note that the extinction intensity change in the case of GNR-anti-TnT-PNIPAAM is higher that the GNR-anti-TnT11. The enhanced extinction intensity was due to a change in the effective refractive index of the medium, which is mainly caused by the PNIPAAM of GNR-anti-TnT-PNIPAAM conjugates. A sharp change (rapid fall) in the extinction at a concentration, ln[TnT] equals to around ~15 is very likely attributed to that fact that incident light frequency nearly matches to the collective plasmon oscillations, as determined by the effective dielectric constant of the medium. As mentioned earlier, anti-TnT-PNIPAAM conjugates showed a maximum extinction (longitudinal) at a wavelength (λLSPR) ~831 nm. The peak in the longitudinal band broadened with increase of TnT concentration, while the transverse band only showed a decrease in extinction intensity with λLSPR shift of 1–2 nm at higher concentrations of TnT. The NIR spectra in the presence of two different representative TnT concentrations (1 and 15 ng/mL) are shown in Fig. 3(b), where λLSPR are observed at ~832 and ~854 nm respectively. The label-free LSPR based nano-immunoassay had a detection limit of 8.4 fg/mL with a response time of 10 sec. at 25 °C. The amplified rectangular area of Fig. 3(b) highlighted as 2(c′) is shown in Fig. 3(c) and the λLSPR for 1 and 15 ng/mL spectra is indicated by the left and right boundary of the green area, respectively. According to the results shown in Fig. 3(d), indicate the comparable values of peak shifting obtained with respect to the mentioned concentrations, which support the dissolution effects of TnT in a biological concentration. A red-shift in the λLSPR was noticed with the increase of the TnT concentration35. It is believed that higher TnT concentration changes effective material index eventually leading to a red shift in excitation peak.

View Article: PubMed Central - PubMed

ABSTRACT

Regeneration of immunosensors is a longstanding challenge. We have developed a re-usable troponin-T (TnT) immunoassay based on localised surface plasmon resonance (LSPR) at gold nanorods (GNR). Thermosensitive poly(N-isopropylacrylamide) (PNIPAAM) was functionalised with anti-TnT to control the affinity interaction with TnT. The LSPR was extremely sensitive to the dielectric constant of the surrounding medium as modulated by antigen binding after 20 min incubation at 37 °C. Computational modelling incorporating molecular docking, molecular dynamics and free energy calculations was used to elucidate the interactions between the various subsystems namely, IgG-antibody (c.f., anti-TnT), PNIPAAM and/or TnT. This study demonstrates a remarkable temperature dependent immuno-interaction due to changes in the PNIPAAM secondary structures, i.e., globular and coil, at above or below the lower critical solution temperature (LCST). A series of concentrations of TnT were measured by correlating the λLSPR shift with relative changes in extinction intensity at the distinct plasmonic maximum (i.e., 832 nm). The magnitude of the red shift in λLSPR was nearly linear with increasing concentration of TnT, over the range 7.6 × 10−15 to 9.1 × 10−4 g/mL. The LSPR based nano-immunoassay could be simply regenerated by switching the polymer conformation and creating a gradient of microenvironments between the two states with a modest change in temperature.

No MeSH data available.