A superior bright NIR luminescent nanoparticle preparation and indicating calcium signaling detection in cells and small animals

Abstract

Background: Near-field fluorescence (NFF) effects were employed to develop a novel near-infrared (NIR) luminescent nanoparticle (LNP) with superior brightness. The LNP is used as imaging contrast agent for cellular and small animal imaging and furthermore suggested to use for detecting voltage-sensitive calcium in living cells and animals with high sensitivity.

Results: NIR Indocyanine green (ICG) dye was conjugated with human serum albumin (HSA) followed by covalently binding to gold nanorod (AuNR). The AuNR displayed dual plasmons from transverse and longitudinal axis, and the longitudinal plasmon was localized at the NIR region which could efficiently couple with the excitation and emission of ICG dye leading to a largely enhanced NFF. The enhancement factor was measured to be about 16-fold using both ensemble and single nanoparticle spectral methods. As an imaging contrast agent, the ICG-HSA-Au complex (abbreviate as ICG-Au) was conjugated on HeLa cells and fluorescence cell images were recorded on a time-resolved confocal microscope. The emission signals of ICG-Au complexes were distinctly resolved as the individual spots that were observed over the cellular backgrounds due to their strong brightness as well as shortened lifetime. The LNPs were also tested to have a low cytotoxicity. The ICG-Au complexes were injected below the skin surface of mouse showing emission spots 5-fold brighter than those from the same amount of free ICG-HSA conjugates.

Conclusions: Based on the observations in this research, the excitation and emission of NIR ICG dyes were found to be able to sufficiently couple with the longitudinal plasmon of AuNRs leading to a largely enhanced NFF. Using the LNP with super-brightness as a contrast agent, the ICG-Au complex could be resolved from the background in the cell and small animal imaging. The novel NIR LNP has also a great potential for detection of voltage-gated calcium concentration in the cell and living animal with a high sensitivity.

Keywords: Dual-mode plasmons; Fluorescence imaging; Gold nanorod (AUNR); Imaging contrast agent; Indocyanine green (ICG); Luminescent nanoparticle (LNP); Near-field fluorescence (NFF).

Fig. 1

a Absorption spectra of AuNRs as CTAB-coated, PEG-coated, and ICG–HSA conjugate-bound in a 10 mM PBS buffer solution. b Ensemble emission spectra from the ICG dyes as free, conjugates in HSA, and complexes with AuNRs in a 10 mM PBS buffer solution

Fig. 2

TEM images of (a) CTAB-AuNRs and (b) ICG-Au complexes

Fig. 3

Upper panels represent the emission imaging from (a) ICG-Au complexes and (b) ICG–HSA conjugates. Diagrams are 5 × 5 µm and resolutions are 100 × 100 pixels with an integration of 0.6 ms/pixel. Bottom panels represent fluorescence images from the cells conjugated with (c) ICG-Au complexes and (d) ICG–HSA conjugates. Diagrams are 50 × 50 µm and resolutions are 100 × 100 pixel with an integration of 0.6 ms/pixel. The samples were excited with a 640 nm laser. Note the different intensity scales. The images of a and c were collected with a laser power 10-fold less than the images of b and d

Fig. 4

Histogram distributions of single sots of (a) emission intensities and (b) lifetimes from the ICG–HSA conjugates as free and bound on the AuNRs

Fig. 5

The panel of cell images of live HeLa cells stained with calcein AM without (a) the Au nanoparticle and (b) in the presence of 3 nM of Au nanoparticles. The images of calcein stained cells (a) and (b) were acquired after 24 h of nanoparticle treatments upon excitation with a 443 laser diode and at bandpass filter 514/30 nm. The images of c and d represent the autofluorescence of cells without (a) the Au nanoparticle and (b) in the presence of 3 nM of Au nanoparticles after 24 h. The autofluorescence images of cells were collected upon an excitation at 640 nm and with a longpass filter of 655 nm. Cells with brighter autofluorescence in c and d are classified as dead. e represents rates of viable cells in the presence of 0.3 and 3 nM in the cell medium as well as in the absence of Au nanoparticle as the control at time interval = 0.5, 2, 12, 24 h

Fig. 6

In-vivo fluorescence tomography images of mice using the ICG-Au complexes as contrast agent injected below the skin of mice. The images were collected on a Xenogen IVIS-200 small animal tomography system with a bandpass filter from 665 to 695 nm for the background, a filter from 710 to 760 on the excitation side, and a filter from 810 to 875 nm on the emission side. A 750 nm laser was used as the excitation source. Total photon flux (photons/s) was calculated and corrected for tissue depth by spectral imaging using Living Image 3.0 software (Xenogen). The left image (a) was collected on a control mice and the right image (b) was collected by injection with the ICG-Au complex, ICG–HSA conjugate, or a blank PBS buffer solution with the same volume on the same mice

Fig. 7

Emission spectral change of ICG-Au complex in 10 mM PBS buffer solution before and after a NaCN treatment