a-label-free-technique-for-spatio-temporal-imaging-single-cell

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Naval Research Laboratory

Today's biosensors and drug delivery devices are increasingly incorporating lithographically patterned circuitry that is placed within microns of the biological molecules to be detected or released. Elevated temperatures due to Joule heating from the underlying circuitry cannot only reduce device performance, but also alter the biological activity of such molecules (i.e. binding, enzymatic, folding). As a consequence, biochip design and characterization will increasingly require local measurements of the temperature and temperature gradients on the biofunctionalized surface. We have developed a technique to address this challenge based on the use of DNA molecular beacons as a nanoscale temperature probe. The surface of fused-silica chips with lithographically patterned, current-carrying gold rings have been functionalized with a layer of molecular beacons. We utilize the temperature dependence of the molecular beacons to calibrate the temperature at the center of the rings as a function of applied current from 25 to 50 degrees C. The fluorescent images of the rings reveal the extent of heating to the surrounding chip due to the applied current while resolving temperature gradients over length scales of less than 500nm. Finite element analysis and analytic calculations of the distribution of heat in the vicinity of the current-carrying rings agree well with the experimental results. Thus, molecular beacons are shown to be a viable tool for temperature calibration of micron-sized circuitry and the visualization of submicron temperature gradients.

The use of DNA molecular beacons as nanoscale temperature probes for microchip-based biosensors.

Magnetic nanoparticles are used throughout biology for applications from targeted drug and gene delivery to the labeling of cells. These nanoparticles typically react with the biological medium to which they are introduced, resulting in a diminished magnetic moment. The rate at which their magnetic moment is diminished limits their utility for targeting and can signal the unintended release of surface-functionalized biomolecules. A foreknowledge of the time-dependent degradation of the magnetic moment in a given medium can aid in the selection of the optimal buffering solution and in the prediction of a reasonable experimental time frame. With this goal in mind, we have developed a SQUID magnetometer based methodology for measuring the saturation magnetic moment of nanoparticles in real time while immersed in a biological medium. Measurements on Co and Ni nanowires in a variety of commonly used buffered salines demonstrated that the technique has the dynamic range and sensitivity to detect the rapid reduction in moment due to active corrosion as well as much more subtle changes from the formation of a passivating surface oxide layer. In order to correlate the magnetic moment reductions to these specific chemical processes, samples were additionally characterized using x-ray photoelectron spectroscopy, inductively coupled plasma spectroscopy and scanning electron microscopy. The most reactive buffers studied were found to be phosphate and carbonate based, which caused active corrosion of the Co nanowires but only a comparatively slow passivation of the Ni nanowires by oxidation.

Magnetic moment degradation of nanowires in biological media: real-time monitoring with SQUID magnetometry.

The pH-dependent binding affinity of either avidin or streptavidin for iminobiotin has been utilized in studies ranging from affinity binding chromatography to dynamic force spectroscopy. Regardless of which protein is used, the logarithmic dependence of the equilibrium dissociation constant (K(d)) on pH is assumed conserved. However a discrepancy has emerged from a number of studies which have shown the binding affinity of streptavidin for iminobiotin in solution to be unexpectedly low, with the K(d) at values usually associated with non-specific binding even at strongly basic pH levels. In this work we have utilized a Bodipy fluorescent conjugate of avidin and an Oregon Green fluorescent conjugate of streptavidin to determine the K(d) of the complexes in solution in the pH range of 7.0 to 10.7. The study was made possible by the remarkable fluorescent enhancement of the two fluorescent conjugates (greater than 10 fold) upon saturation with iminobiotin. The streptavidin-iminobiotin interaction exhibited almost no pH dependence over the range studied, with K(d) consistently on the order of 10(-5) M. In contrast, under identical experimental conditions the avidin-iminobiotin interaction exhibited the expected logarithmic dependence on pH. We discuss the possible origins for why these two closely related proteins would diverge in their binding affinities for iminobiotin as a function of pH.

Iminobiotin binding induces large fluorescent enhancements in avidin and streptavidin fluorescent conjugates and exhibits diverging pH-dependent binding affinities.

A new quantitative analysis methodology for localized surface plasmon resonance (LSPR) biosensing which determines surface-receptor fractional occupancy, as well as an LSPR imaging technique for the spatiotemporal mapping of binding events, is presented. Electron beam nanolithography was used to fabricate 20 × 20 arrays of gold nanostructures atop glass coverslips. A single biotinylated array was used to measure the association kinetics of neutravidin to the surface by spectroscopically determining the fractional occupancy as a function of time. By regenerating the same array, a reliable comparison of the kinetics could be made between control samples and neutravidin concentrations ranging from 1 μM to 50 nM. CCD-based imagery of the array, taken simultaneously with the spectroscopic measurements, reveals the binding of neutravidin to the surface as manifested by enhanced scattering over the majority of the resonance peak. The temporal resolution of the LSPR imaging technique was 200 ms and the spatial resolution was 8 μm(2).

A new methodology for quantitative LSPR biosensing and imaging.

Localized surface plasmon resonance (LSPR) imaging has the potential to map complex spatio-temporal variations in analyte concentration, such as those produced by protein secretions from live cells. A fundamental roadblock to the realization of such applications is the challenge of calibrating a nanoscale sensor for quantitative analysis. Here, we introduce a new, to our knowledge, LSPR imaging and analysis technique that enables the calibration of hundreds of individual gold nanostructures in parallel. The calibration allowed us to map the fractional occupancy of surface-bound receptors at individual nanostructures with nanomolar sensitivity and a temporal resolution of 225 ms. As a demonstration of the technique's applicability to molecular and cell biology, the calibrated array was used for the quantitative LSPR imaging of anti-c-myc antibodies harvested from a cultured 9E10 hybridoma cell line without the need for further purification or processing.

Quantitative LSPR imaging for biosensing with single nanostructure resolution.

Protein secretions from individual cells create spatially and temporally varying concentration profiles in the extracellular environment, which guide a wide range of biological processes such as wound healing and angiogenesis. Fluorescent and colorimetric probes for the detection of single cell secretions have time resolutions that range from hours to days, and as a result, little is known about how individual cells may alter their protein secretion rates on the timescale of minutes or seconds. Here, we present a label-free technique based upon nanoplasmonic imaging, which enabled the measurement of individual cell secretions in real time. When applied to the detection of antibody secretions from single hybridoma cells, the enhanced time resolution revealed two modes of secretion: one in which the cell secreted continuously and another in which antibodies were released in concentrated bursts that coincided with minute-long morphological contractions of the cell. From the continuous secretion measurements we determined the local concentration of antibodies at the sensing array closest to the cell and from the bursts we estimated the diffusion constant of the secreted antibodies through the extracellular media. The design also incorporates transmitted light and fluorescence microscopy capabilities for monitoring cellular morphological changes and intracellular fluorescent labels. We anticipate that this technique can be adapted as a general tool for the quantitative study of paracrine signaling in both adherent and nonadherent cell lines.

Jeff M. Byers

Materials Science and Technology

Naval Research Laboratory

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions