Gold Nanourchins Improve Virus Targeting and Plasmonic Coupling for Virus Diagnosis on a Smartphone Platform.

Point-of-care detection of pathogens is critical to monitor and combat viral infections. The plasmonic coupling assay (PCA) is a homogeneous assay and allows rapid, one-step, and colorimetric detection of intact viruses. However, PCA lacks sufficient sensitivity, necessitating further mechanistic studies to improve the detection performance of PCA. Here, we demonstrate that gold nanourchins (AuNUs) provide significantly improved colorimetric detection of viruses in PCA. Using respiratory syncytial virus (RSV) as a target, we demonstrate that the AuNU-based PCA achieves a detection limit of 1400 PFU/mL, or 17 genome equivalent copies/µL. Mechanistic studies suggest that the improved detection sensitivity arises from the higher virus-binding capability and stronger plasmonic coupling at long distances (∼10 nm) by AuNU probes. Furthermore, we demonstrate the virus detection with a portable smartphone-based spectrometer using RSV-spiked nasal swab clinical samples. Our study uncovers important mechanisms for the sensitive detection of intact viruses in PCA and provides a potential toolkit at the point of care.

Ultrabright Fluorescent Silica Nanoparticles for Dual pH and Temperature Measurements.

The mesoporous nature of silica nanoparticles provides a novel platform for the development of ultrabright fluorescent particles, which have organic molecular fluorescent dyes physically encapsulated inside the silica pores. The close proximity of the dye molecules, which is possible without fluorescence quenching, gives an advantage of building sensors using FRET coupling between the encapsulated dye molecules. Here we present the use of this approach to demonstrate the assembly of ultrabright fluorescent ratiometric sensors capable of simultaneous acidity (pH) and temperature measurements. FRET pairs of the temperature-responsive, pH-sensitive and reference dyes are physically encapsulated inside the silica matrix of ~50 nm particles. We demonstrate that the particles can be used to measure both the temperature in the biologically relevant range (20 to 50 °C) and pH within 4 to 7 range with the error (mean absolute deviation) of 0.54 °C and 0.09, respectively. Stability of the sensor is demonstrated. The sensitivity of the sensor ranges within 0.2-3% °C-1 for the measurements of temperature and 2-6% pH-1 for acidity.

In Vivo Targeting of Xenografted Human Cancer Cells with Functionalized Fluorescent Silica Nanoparticles in Zebrafish.

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal models are required for bridging the nanotechnology to its biomedical application. The mouse represents the traditional animal model for preclinical testing; however, mice are relatively expensive to keep and have long experimental cycles due to the limited progeny from each mother. The zebrafish has emerged as a powerful model system for developmental and biomedical research, including cancer research. In particular, due to its optical transparency and rapid development, zebrafish embryos are well suited for real-time in vivo monitoring of the behavior of cancer cells and their interactions with their microenvironment. This method was developed to sequentially introduce human cancer cells and functionalized nanoparticles in transparent Casper zebrafish embryos and monitor in vivo recognition and targeting of the cancer cells by nanoparticles in real time. This optimized protocol shows that fluorescently labeled nanoparticles, which are functionalized with folate groups, can specifically recognize and target metastatic human cervical epithelial cancer cells labeled with a different fluorochrome. The recognition and targeting process can occur as early as 30 min postinjection of the nanoparticles tested. The whole experiment only requires the breeding of a few pairs of adult fish and takes less than 4 days to complete. Moreover, zebrafish embryos lack a functional adaptive immune system, allowing the engraftment of a wide range of human cancer cells. Hence, the utility of the protocol described here enables the testing of nanoparticles on various types of human cancer cells, facilitating the selection of optimal nanoparticles in each specific cancer context for future testing in mammals and the clinic.

Ultrabright Fluorescent Silica Nanoparticles for Multiplexed Detection.

Fluorescent tagging is a popular method in biomedical research. Using multiple taggants of different but resolvable fluorescent spectra simultaneously (multiplexing), it is possible to obtain more comprehensive and faster information about various biochemical reactions and diseases, for example, in the method of flow cytometry. Here we report on a first demonstration of the synthesis of ultrabright fluorescent silica nanoporous nanoparticles (Star-dots), which have a large number of complex fluorescence spectra suitable for multiplexed applications. The spectra are obtained via simple physical mixing of different commercially available fluorescent dyes in a synthesizing bath. The resulting particles contain dye molecules encapsulated inside of cylindrical nanochannels of the silica matrix. The distance between the dye molecules is sufficiently small to attain Forster resonance energy transfer (FRET) coupling within a portion of the encapsulated dye molecules. As a result, one can have particles of multiple spectra that can be excited with just one wavelength. We show this for the mixing of five, three, and two dyes. Furthermore, the dyes can be mixed inside of particles in different proportions. This brings another dimension in the complexity of the obtained spectra and makes the number of different resolvable spectra practically unlimited. We demonstrate that the spectra obtained by different mixing of just two dyes inside of each particle can be easily distinguished by using a linear decomposition method. As a practical example, the errors of demultiplexing are measured when sets of a hundred particles are used for tagging.

Ultrabright fluorescent silica nanoparticles for in vivo targeting of xenografted human tumors and cancer cells in zebrafish.

New ultrabright fluorescent silica nanoparticles capable of the fast targeting of epithelial tumors in vivo are presented. The as-synthesized folate-functionalized ultrabright particles of 30-40 nm are 230 times brighter than quantum dots (QD450) and 50% brighter than the polymer dots with similar spectra (excitation 365 nm and emission 486 nm). To decrease non-specific targeting, particles are coated with polyethylene glycol (PEG). We demonstrate the in vivo targeting of xenographic human cervical epithelial tumors (HeLa cells) using zebrafish as a model system. The particles target tumors (and probably even individual HeLa cells) as small as 10-20 microns within 20-30 minutes after blood injection. To demonstrate the advantages of ultrabrightness, we repeated the experiments with similar but 200× less bright particles. Compared to those, ultrabright particles showed ∼3× faster tumor detection and ∼2× higher relative fluorescent contrast of tumors/cancer cells.

Magnetic field directed fabrication of conducting polymer nanowires.

The self-assembly of nanoparticles is an efficient and precise method to fabricate nanoscale devices. By manipulating iron oxide nanoparticles in suspension with an external field to form magnetically directed linear assemblies, we demonstrate the feasibility of using this structure to template the synthesis of PEDOT:PSS conducting polymer nanowires in suspension. Furthermore these conducting wires can be assembled on interdigitated electrodes to form an array of conducting nanowires.