Gold Nanocluster Containing Polymeric Microcapsules for Intracellular Ratiometric Fluorescence Biosensing

Abstract

A new approach to sensing and imaging hydrogen peroxide (H₂O₂) was developed using microcapsule-based dual-emission ratiometric luminescent biosensors. Bovine serum albumin-capped gold nanoclusters (BSA-AuNCs) sensitive to H₂O₂ were coencapsulated with insensitive FluoSpheres (FSs) within polymeric capsules fabricated via the layer-by-layer method. Under single-wavelength excitation, the microcapsule-based biosensors exhibited emission bands at ∼516 and ∼682 nm resulting from the FSs and BSA-AuNCs, respectively. The polyelectrolyte multilayers lining the microcapsules were effective in protecting BSA-AuNCs from the degradation catalyzed by proteases (chymotrypsin, trypsin, papain, and proteinase K) and subsequent luminescent quenching, overcoming a key limitation of prior BSA-AuNC-based sensing systems. The luminescent response of the sensors was also found to be independent of local changes in pH (5-9). Quenching of the AuNCs in the presence of H₂O₂ enabled the spectroscopic quantification and imaging of changes in H₂O₂ concentration from 0 to 1 mM. The microcapsule sensors were easily phagocytized by murine macrophage cells (RAW 264.7), were effective as intracellular H₂O₂ imaging probes, and were successfully used to detect local release of H₂O₂ in response to an external chemical stimulus.

Figure 1

(A) Normalized emission spectra of FSs (green dashed line), BSA-AuNC (red dashed line), and microcapsules containing both FSs and BSA-AuNC (pink line), (Inset) photographs of luminescent FSs, BSA-AuNC, and microcapsules containing both FSs and BSA-AuNC suspended in solution under UV illumination, (B) plots of R/R⁰ of nonencapsulated BSA-AuNC (dark gray stripe) and encapsulated BSA-AuNC (dark gray solid) in the presence of proteases. Here R and R⁰ represent BSA-AuNC luminescence intensities in the presence and absence of proteases respectively. Error bars represent 95% confidence intervals for three separate batches of sensors.

Scheme 1. (A) Microcapsule-Based Hydrogen Peroxide Sensor, (B) BSA-AuNC, (C) FS, (D) Microcapsule Sensors Incubated with Macrophages, (E) Microcapsule Sensors Being Engulfed by Macrophages, and (F) Microcapsule Sensors Ingested by Macrophages

Figure 2

Ratiometric response of microcapsule sensors at different pH, normalized to (A) BSA-AuNC peak (682 nm) and (B) FSs peak (516 nm). Error bars represent 95% confidence intervals for three separate batches of microcapsule sensors.

Figure 3

(A) Emission spectra of microcapsules containing both FSs and BSA-AuNC to 0 μM (black), 20 μM (purple), 40 μM (blue), 60 μM (maroon), 80 μM (navy blue), 100 μM (orange), 200 μM (bright red), 400 μM (pink), 600 μM (green), 800 μM (red), and 1000 μM hydrogen peroxide (light blue); mean ratiometric response of microcapsules containing both FSs and BSA-AuNC to varying concentrations of hydrogen peroxide (B) obtained using a microplate spectrophotometer and (C) obtained using ratiometric images of microcapsules. Confocal, ratiometric fluorescence images of microcapsules containing both FSs and BSA-AuNC at (D) 0 μM, (E) 400 μM, and (F) 1000 μM hydrogen peroxide. Pseudocolored images represent the ratio of emission intensities collected using a 510–540 nm band-pass filter and a 633 nm long-pass filter, when excited at 445 nm. The scale bars correspond to 20 μm. The error bars represent 95% confidence intervals for at least three separate samples of microcapsule sensors.

Figure 4

Confocal fluorescence images of RAW 264.7 macrophage cells. The pseudocolored images represent the ratio of emission intensities collected using a 510–540 nm band-pass filter and a 633 nm long-pass filter, when excited at 445 nm. (A) Cells incubated with microcapsules for 1 h at 37 °C, (B) microcapsule-loaded cells after PMA (2 μg/mL) exposure for 30 min at 37 °C, and (C) microcapsule-loaded cells after H₂O₂ (500 μM) exposure for 30 min at 37 °C. (D–F) DIC images of the cells in (A)–(C), respectively. The scale bars correspond to 30 μm. (G) Ratiometric response of extracellular (dark gray stripes) and intracellular (dark gray solid) microcapsule sensors. The error bars represent 95% confidence intervals for three separate batches of microcapsule sensors.