Multiple sharp‐edged gold nanostars were efficiently assembled on nanopipette tips through electrostatic interactions for use as a potent intracellular hypoxia‐sensing Raman probe. Colloidal stability and surface immobilization were checked using scanning electron microscopy, light scattering, and zeta potential measurements. Site‐specific intracellular hypoxia levels can be estimated in vitro and in vivo using Raman lancets (RL). Distinct Raman spectral changes for the nitro‐(NO2) functional group of the redox marker 4‐nitrothiophenol (4NTP) can be quantified according to the intracellular oxygen (O2) content, ranging from 1 % to 10 %. Redox potential changes in mitochondrial respiration were also examined through serial injections of inhibitors. 3D‐cultured cells and in vivo tests were used to validate our method, and its application in the assessment of the aggressiveness of cancer cells by differentiating spectral changes between malignant and benign cells was demonstrated.

To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.

No abstract

We demonstrate a label-free protein detection and separation technology for real-time monitoring of proteins in micro/nanofluidic channels, confined surface plasmon resonance imaging (confined-SPRi). This was achieved by fabricating ultrathin fluidic channels (500 nm high, 500 microm wide) directly on top of a specialized SPRi sensor surface. In this way, SPRi is uniquely used to detect proteins deep into the fluidic channel while maintaining high lateral accuracy of separated products. The channel fluid and proteins were driven electrokinetically under an external electric field. For this to occur, the metallic SPR sensor (46 nm of Au on 2 nm of Cr) was segmented into an array of squares (each 200 microm x 200 microm in size and spaced 8 microm apart) and coated with 30 nm of CYTOP polymer. In this work, we track label-free protein separation in real time through a simple cross-junction fluidic device with an 8-mm separation channel length under 30 V/cm electric field strength.

Gold nanoclusters are emerging as new materials for biomedical applications because of promises offered by their ultrasmall size and excellent biocompatibility. Here, the synthesis and optical and biological characterizations of a highly luminescent folate-functionalized Au cluster (Au -FA) are reported. The Au -FA clusters are synthesized by functionalizing the surface of Au (SG) clusters, where SG is glutathione, with benzyl chloroformate and folate. The functionalized clusters are highly water-soluble and exhibit remarkably bright luminescence with a quantum yield of 42%, significantly higher than any other water-soluble gold clusters protected with thiolate ligands. The folate groups conjugated to the gold cluster give rise to additional luminescence enhancement by energy transfer sensitization. The brightness of Au -FA is found to be 4.77 mM cm , nearly 8-fold brighter than that of Au (SG) . Further biological characterizations have revealed that the Au -FA clusters are well-suited for bioimaging. The Au -FA clusters exhibit excellent photostability and low toxicity; nearly 80% cell viability at 1000 ppm of the cluster. Additionally, the Au -FA clusters show target specificity to folate-receptor positive cells. Finally, the time-course in vivo luminescence images of intravenous-injected mice show that the Au -FA clusters are renal-clearable, leaving only 8% of them remained in the body after 24 h post-injection.