Chapter 1 Introduction
2.4 Conclusion
Figure 2.16: Comparing the mouse hematopoetic stem cell uptake of cKit conju- gated barium titanate nanoprobes (denoted as F) to the control BT-OH nanoprobes (denoted as C), enhancement in the uptake is observed. The cell surface coverage of 18% as compared to the 11% for the control BT-OH probes. A two-tailed T-test with unequal variance conrms that the BT-cKit and BT-OH are dierently uptaken by the cells with high condence (p-value < 10-4).
attached: 4008 cells counted total in the control BT-OH case and 6756 cells total for the surface functionalized BT-cKit case. Two-tailed T-test with unequal variance, determined a signicant p-value that is <<10-4with an enhanced cell surface coverage of 18% in the case of BT-cKit, as opposed to the 11% nonspecic cell surface coverage for the control (gure 2.16).
functionalization steps, including EDC/NHS coupling to carboxylic acid functional groups and hydrazine linkage to aldehyde groups on oxidized sugar residues on an- tibodies, allowed barium titanate SHG nanoprobes to be coated with a variety of ligands for both nontargeted and targeted functionalizations. The covalently bound ligands were stable over a course of weeks and in dierent environments, as conrmed by repeated colocalization of SHG and uorescent signal, when appropriate, or by FT-IR analysis for BT-NH2 samples. The use of BT SHG probes in in vivo allows for long-term imaging of stable, nontoxic, biocompatible probes that are not limited by dye saturation, dye bleaching or blinking.
The procedures presented in this chapter are not exhaustive and can be modied for alternative functionalities or model organisms. Use of alternative biocompati- ble polymerssuch as the poly(acrylic acid) (PAA) polymer [56]can provide an even more widespread binding to the BT surface than monofunctional PEG, enabling greater surface coverage. Additionally, utilizing commercially available heterofunc- tional PEG reagents, which contain dierent functional groups on their ends, SHG nanoprobe surfaces could be specically targeted. This could be achieved by careful selection of functional groups that posses a high anity to the surface and surface exposed functional, while the other functional end on the PEG polymer would be utilized for specic targeting of biological sructures.
The biotin functionalization presented here served as a straightforward means of gauging the success of the modication procedure by means of colocalization of AlexaFluor488 uorescent and BT SHG signal. However, the same biotin-BT probe can be directly used for targeted imaging. Biotin has a high anity for avidin and steptavidin (KD∼10-15) proteins and the biotin-BT probes could be used for sensing applications, among others [75, 76].
Barium titanate SHG nanoprobes have also been functionalized with uorescent dyes, corroles. Corroles are small molecules which can be synthesized with a variety of functional groups attached, and thus tuned for dierent photophysical properties.
Corroles are especially interesting for the synthesis of novel imaging probes that can interact with NIR light and emit photons in the NIR range as well. By careful design,
the SHG nanoprobes will supply the visible light, needed for the corrole uorescence excitation. The relationship of this SHG enhanced uorescence with respect to the distance between the BT nanoparticle and the corrole can be investigated as a function of the length of the intermediate linker between the two imaging probes. Therefore, utilizing corroles with dierent functionalities, one can investigate the SHG enhance- ment of the uorescent signal as well as the photophysical properties of a variety of dierent BT-corrole probes.
In general, the major limitation of the SHG nanoprobes in biological imaging ap- plications is straightforward: in contrast to uorescent proteins, these probes cannot be used in applications requiring genetically encoded labeling. Additionally, since commercially available ∼100-200 nm BT nanocrystals are the starting material for functionalization in this protocol, applications for single molecule analysis or ecient cell or protein targeting are limited, even when extending the protocol to targeted labeling such as with the biotin linking chemistry. This restriction will be lifted once ecient synthesis of monodisperse, tetragonal BT SHG nanoprobes is accomplished with sizes comparable to currently used uorescent probes (less than 10 nm). Current established BT synthesis protocols in the desired size range only result in cubic (i.e.
centrosymmetric) crystal structures due to quantum connement eects [77].
Monodispersity of SHG nanoprobes is desired for a number of reasons. Polydis- perse nanoparticles have a wide distribution of sizes which in general limits their application in biological systems. It has been demonstrated that the distribution of nanoparticles in the body and their interaction with cells is greatly inuenced by the size of the nanoparticles, as well as their targeting ligand content [51]. In addi- tion, monodisperse particles posses uniform physical and chemical properties, which enables their easier characterization, functionalization, and ultimately application.
By controlling the size, and thus the properties of the particles, many interesting studies can be conducted, such as determining the dependence of the SHG signal on the size of the probe. Currently reported values of the second order susceptibilities for dierent SHG nanoprobes, have been normalization over the average nanoparti- cle volumes, introducing calculation errors due to the broad size distributions [78].
Therefore, a fundamental study on the SHG eciency in monodisperse nanoparticle samples would allow greater understanding of their nonlinear optical properties. This would allow for more sensitive multispectral imaging with SHG nanoprobes, as the intensity of the SHG signal would be better resolved [21].