Stefan Wilhelm

Optical sensing of the ionic strength using photonic crystals in a hydrogel matrix.

Monodisperse, highly negatively charged, cross-linked polystyrene nanoparticles with diameters between 80 and 120 nm have been incorporated into a polyacrylamide hydrogel, where they display an iridescent color that conventionally is attributed to the so-called photonic crystal effect. The film is of red color if placed in plain water but turns to green in the presence of a 1 mM solution of an electrolyte such as sodium chloride and to purple in 100 mM solutions of electrolytes. Quantitative reflection spectroscopy was performed at various wavelengths and resulted in plots of reflected light wavelength versus ionic strength (IS) that are almost linear in the logarithmic concentration range from 5 × 10(-5) to 10(-2) mol·L(-1). We show that such films are capable of monitoring the IS of aqueous solutions in the pH range from 5 to 9. We also show that, in addition to visual and instrumental readout, the sensor films can be analyzed with a digital camera at fixed angle. The digital images were separated into their red, green, and blue channels and analyzed. The red channel was found to be best suited for determination of the IS and resulted in calibration plots that are comparable if not better than those obtained by reflectometry.

Multicolor Upconversion Nanoparticles for Protein Conjugation

We describe the preparation of monodisperse, lanthanide-doped hexagonal-phase NaYF4 upconverting luminescent nanoparticles for protein conjugation. Their core was coated with a silica shell which then was modified with a poly(ethylene glycol) spacer and N-hydroxysuccinimide ester groups. The nanoparticles were characterized by transmission electron microscopy, Raman spectroscopy, X-ray diffraction, and dynamic light scattering. The N-hydroxysuccinimide ester functionalization renders them highly reactive towards amine nucleophiles (e.g., proteins). We show that such particles can be conjugated to proteins. The protein-reactive UCLNPs and their conjugates to streptavidin and bovine serum albumin display multicolor emissions upon 980-nm continuous wave laser excitation. Surface plasmon resonance studies were carried out to prove bioconjugation and to compare the affinity of the particles for proteins immobilized on a thin gold film.

Spectrally matched upconverting luminescent nanoparticles for monitoring enzymatic reactions.

We report on upconverting luminescent nanoparticles (UCLNPs) that are spectrally tuned such that their emission matches the absorption bands of the two most important species associated with enzymatic redox reactions. The core-shell UCLNPs consist of a β-NaYF4 core doped with Yb(3+)/Tm(3+) ions and a shell of pure β-NaYF4. Upon 980 nm excitation, they display emission bands peaking at 360 and 475 nm, which is a perfect match to the absorption bands of the enzyme cosubstrate NADH and the coenzyme FAD, respectively. By exploiting these spectral overlaps, we have designed fluorescent detection schemes for NADH and FAD that are based on the modulation of the emission intensities of UCLNPs by FAD and NADH via an inner filter effect.

Three-Dimensional Optical Mapping of Nanoparticle Distribution in Intact Tissues

The role of tissue architecture in mediating nanoparticle transport, targeting, and biological effects is unknown due to the lack of tools for imaging nanomaterials in whole organs. Here, we developed a rapid optical mapping technique to image nanomaterials in intact organs ex vivo and in three-dimensions (3D). We engineered a high-throughput electrophoretic flow device to simultaneously transform up to 48 tissues into optically transparent structures, allowing subcellular imaging of nanomaterials more than 1 mm deep into tissues which is 25-fold greater than current techniques. A key finding is that nanomaterials can be retained in the processed tissue by chemical cross-linking of surface adsorbed serum proteins to the tissue matrix, which enables nanomaterials to be imaged with respect to cells, blood vessels, and other structures. We developed a computational algorithm to analyze and quantitatively map nanomaterial distribution. This method can be universally applied to visualize the distribution and interactions of materials in whole tissues and animals including such applications as the imaging of nanomaterials, tissue engineered constructs, and biosensors within their intact biological environment.

Enzyme-induced modulation of the emission of upconverting nanoparticles: Towards a new sensing scheme for glucose

A new approach for the design of a fluorometric biosensor for continuous monitoring of glucose levels in biological samples based on near-infrared (NIR) excitation is described. The sensor combines the fluorescence of the enzyme glucose oxidase (GOx) chemically modified with a fluorescein derivative (FS) and the luminescent properties of upconverting luminescent nanoparticles (UCLNPs). Both, the chemically modified enzyme (GOx-FS) and the UCLNPs are immobilized in a poly(acrylamide) film as a physical support. The excitation of the UCLNPs with NIR light is of major advantage, since fluorescence background from the matrix is minimized. The upconverted luminescence is used to excite GOx-FS, which undergoes a change in the fluorescence intensity during the enzymatic reaction with glucose. The sensor comprises sufficient stability and covers the physiological range of glucose levels in blood. Furthermore, in a proof of principle experiment, the sensor system responds linearly to glucose concentrations in the range from 3.3 to 16.6 mM in flow injection analysis mode.

Perspectives for Upconverting Nanoparticles

Upconverting nanoparticles (UCNPs) are inorganic crystalline nanomaterials that can convert near-infrared (NIR) excitation light into visible and ultraviolet emission light. Excitation with NIR light minimizes autofluorescence background and enables deeper penetration into biological samples due to reduced light scattering. Although these optical features make UCNPs promising candidates for bioanalytical and biomedical applications, the performance of UCNPs is compromised by their low optical brightness. Research in the field of UCNP technology focuses on strategies to boost upconversion luminescence brightness and efficiencies. In this Perspective, I discuss challenges associated with the use of UCNPs and provide a 10-year proposed strategic plan to enable translation of UCNP technology from the academic stage into real world products and applications.

Three-Dimensional Imaging of Transparent Tissues via Metal Nanoparticle Labeling

Chemical probes are key components of the bioimaging toolbox, as they label biomolecules in cells and tissues. The new challenge in bioimaging is to design chemical probes for three-dimensional (3D) tissue imaging. In this work, we discovered that light scattering of metal nanoparticles can provide 3D imaging contrast in intact and transparent tissues. The nanoparticles can act as a template for the chemical growth of a metal layer to further enhance the scattering signal. The use of chemically grown nanoparticles in whole tissues can amplify the scattering to produce a 1.4 million-fold greater photon yield than obtained using common fluorophores. These probes are non-photobleaching and can be used alongside fluorophores without interference. We demonstrated three distinct biomedical applications: (a) molecular imaging of blood vessels, (b) tracking of nanodrug carriers in tumors, and (c) mapping of lesions and immune cells in a multiple sclerosis mouse model. Our strategy establishes a distinct yet complementary set of imaging probes for understanding disease mechanisms in three dimensions.

Exploring Passive Clearing for 3D Optical Imaging of Nanoparticles in Intact Tissues

The three-dimensional (3D) optical imaging of nanoparticle distribution within cells and tissues can provide insights into barriers to nanoparticle transport in vivo. However, this approach requires the preparation of optically transparent samples using harsh chemical and physical methods, which can lead to a significant loss of nanoparticles and decreased sensitivity of subsequent analyses. Here, we investigate the influence of electrophoresis and clearing time on nanoparticle retention within intact tissues and the impact of these factors on the final 3D image quality. Our findings reveal that longer clearing times lead to a loss of nanoparticles but improved transparency of tissues. We discovered that passive clearing improved nanoparticle retention 2-fold compared to results from electrophoretic clearing. Using the passive clearing approach, we were able to observe a small population of nanoparticles undergoing hepatobiliary clearance, which could not be observed in liver tissues that were prepared by electrophoretic clearing. This strategy enables researchers to visualize the interface between nanomaterials and their surrounding biological environment with high sensitivity, which enables quantitative and unbiased analysis for guiding the next generation of nanomedicine designs.

A reagentless enzymatic fluorescent biosensor for glucose based on upconverting glasses, as excitation source, and chemically modified glucose oxidase.

Upon near-infrared excitation Tm(3+)+Yb(3+) doped fluorohafnate glasses present upconversion properties and emit visible light. This property permits to use these glasses (UCG) as excitation sources for fluorescent optical biosensors. Taking this into account, in this work a fluorescent biosensor for glucose determination is designed and evaluated. The biosensor combines the UCG and the fluorescence of the enzyme glucose oxidase chemically modified with a fluorescein derivative (GOx-FS), whose intensity is modified during the enzymatic reaction with glucose. Optical parameters have been optimized and a mathematical model describing the behavior of the analytical signal is suggested. Working in FIA mode, the biosensor responds to glucose concentrations up to, at least, 15mM with a limit of detection of 1.9mM. The biosensor has a minimum lifetime of 9 days and has been applied to glucose determination in drinks. The applicability of the sensor was tested by glucose determination in two fruit juices.