Analysis of Polar Compounds with Ion Pair Reagents

Maria Ada Malvindi, Gabriele Maiorano, Pier Paolo Pompa

Center for Bio-Molecular Nanotechnology, Italian Institute of Technology (IIT) Via Barsanti - 73010 Arnesano(Lecce), Italy

Introduction

Fluorescent nanobeads offer great potential for many applications in both basic and applied research. In the recent past, scientists have been offered a wide range of technical solutions in fluorescence imaging, enabling a significant advancement in fields such as microscopy and diagnostics. However, such optical labels mostly spanned the microscale range and/or suffered from limited optical performance and versatility. Recently, the progress of nanoscience has enabled the fabrication of accurately controlled nanostructures with tailored optical properties, and this is disclosing completely unexplored avenues and exciting possibilities in many research areas. The newly developed fluorescent systems combine very small dimensions (typically <100 nm) with high brightness, photostability, tunable spectral features/surface charge/chemistry, and also biocompatibility. These novel properties allow for new applications where size strongly matters, such as cellular studies, high-resolution microscopies, drug delivery, and sensing.

Possible applications of nanobeads include:

• Fluorescence/Confocal microscopy (including super-resolution techniques)
• Flow cytometry
• Imaging, sensing and diagnostics
• Nanomedicine and nanotoxicology

Fluorescent nanobeads

The bright and stable fluorescence emitters with nanoscale dimensions open many stimulating opportunities in optical imaging. For instance, high resolution applications requiring particle imaging, even at single-particle level, becomes a standard practice rather than a challenging task and can be routinely performed by non-specialized researchers and/or by commercially available instrumentation and devices. This is a fundamental point, as the availability of a fluorescent tag that is highly bright and functionalizable with many (bio)molecules enables the development of many applications in imaging, sensing, and diagnostics. Cellular/tissue imaging can also strongly benefit from the use of fluorescent nanobeads, as their brightness and non-cytotoxicity allow for high resolution intracellular localization and tracking of targets of interests in both fixed and living cells¹. The extreme photostability of the nanobeads in biological environments allow for long-term studies. Even after prolonged irradiation, the nanobeads display minimal or no photobleaching.

The photostability and brightness are advantageous characteristics that can be exploited in super-resolution imaging techniques, such as in Stimulated Emission Depletion (STED) microscopy. STED technique, invented by 2014 Nobel Laureate Stefan W. Hell, allows breaking the diffraction limit of optical microscopes (ca. 200 nm) by selectively deactivating fluorophores, enabling strong enhancement of the lateral resolution (down to 30-40 nm with commercial microscopes) in fluorescence images. This important advancement in imaging has recently led to many outstanding findings in biology and biophysics^2,3. We offer well characterized fluorescent nanobeads (Figure 1) which allow super-resolution STED imaging, permitting a strong push in lateral resolution down to 30 nm. In these applications, the accurate size control and monodispersion provided by this material enables reliable imaging of the real dimensions of the nanobeads. Super-resolved images can also be obtained in the biological environment, including cellular imaging (Figures 2 & 3).

Figure 1.   Left: Multicolor fluorescent nanobeads dispersed in water (emission is available from blue to red/infrared).   Right: Representative TEM image of monodispersed silica nanobeads (Prod No. 797952).

Figure 2. Confocal microscopy image of 120 nm fluorescent nanobeads (Prod No. 797863) on a glass slide (Left), or internalized by A549 cells (Right). A very bright and stable signal is generated by the nanobeads. The biocompatibility and customizable surface chemistry allows successful applications, such as biological imaging and targeted drug delivery.

Figure 3. Confocal and super-resolution gSTED microscopy image of 25 nm fluorescent nanobeads (Prod No. 797901). The high brightness and photostability of the nanobeads easily allows a strong improvement in lateral resolution. In these applications, an accurate control of the particle size is crucial, and our nanobead products offer the best quality enabling superior imaging. The biocompatibility of the nanobeads opens up a wealth of applications in both in-vitro and in-vivo systems.

Another important application of fluorescent nanobeads is represented by flow cytometry. In this  case, the high brightness of the nanobeads makes biological processes and cellular interactions readily detectable by standard instrumentation. Both passive cellular internalization and/or specific targeting can be followed by flow cytometry, even in multicolor formats. The nanobeads can be functionalized with antibodies or aptamers for specific targeting or for diagnostic applications. Fluorescent nanobeads have been employed for sensitive and rapid detection of cancer cells by flow cytometry, obtaining an increased detection sensitivity with respect to standard methods.⁴ In another application, a sensitive and specific strategy for the detection of Staphylococcus aureus was achieved by He et al.⁵, Investigations in nanomedicine and nanotoxicology can strongly benefit from the use of bright nanobeads (Figure 4)

 

Figure 4. Internalization of our fluorescent nanobeads in human primary CD14+ monocytes as probed by flow cytometry. Meaning fluorescence intensity increases as a function of nanobeads concentration.

Summary

In the above applications, the efficiency and versatility of the fluorescent probes are the key parameters. Our Material Science portfolio offers a variety of fluorescent nanobeads, where it is possible to choose the desired size (from 25 to 250 nm) while maintaining an accurate control of particle monodispersion, the fluorescence characteristics (from blue to infrared emission), as well as the surface charge and chemistry (e.g., carboxylic, amino, sulphonic acid groups) to allow for proper biological interactions or specific bioconjugation for targeting. All the fluorescent nanobeads are highly bright and photostable and are characterized by high biocompatibility and sterility.

References

1. Chu Z, Huang Y, Tao Q, Li Q. 2011. Cellular uptake, evolution, and excretion of silica nanoparticles in human cells. Nanoscale. 3(8):3291. https://doi.org/10.1039/c1nr10499c

2. Westphal V, Rizzoli SO, Lauterbach MA, Kamin D, Jahn R, Hell SW. 2008. Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement. Science. 320(5873):246-249. https://doi.org/10.1126/science.1154228

3. Eggeling C, Ringemann C, Medda R, Schwarzmann G, Sandhoff K, Polyakova S, Belov VN, Hein B, von Middendorff C, Schönle A, et al. 2009. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature. 457(7233):1159-1162. https://doi.org/10.1038/nature07596

4. Estévez M-, O?Donoghue MB, Chen X, Tan W. 2009. Highly fluorescent dye-doped silica nanoparticles increase flow cytometry sensitivity for cancer cell monitoring. Nano Res.. 2(6):448-461. https://doi.org/10.1007/s12274-009-9041-8

5. He X, Li Y, He D, Wang K, Shangguan J, Shi H. 2014. Aptamer-Fluorescent Silica Nanoparticles Bioconjugates Based Dual-Color Flow Cytometry for Specific Detection of Staphylococcus aureus. Journal of Biomedical Nanotechnology. 10(7):1359-1368. https://doi.org/10.1166/jbn.2014.1828