New Methods For Biointerface Analysis By Time-Of-Flight Secondary Ion Mass Spectrometry

Surface analysis is different from bulk analysis since the distribution of atoms and electrons at the surface is quite different from that in the bulk. Chemical changes occurring at the liquid surfaces are important in many areas such as biological dynamic process study. Specifically, biological surface analysis includes the characterization of biofilms, cell membrane, biomacromolecules and the study of their biological behaviors. Recent decades have witnessed great advances in surface techniques development, including Time-of-Flight secondary ion mass spectrometry (ToF-SIMS), X-ray photoelectron spectroscopy (XPS), Auger electron spectroscopy (AES), scanning electron microscopy (SEM), etc. However, many of these techniques require vacuum or even high vacuum environments as they are very sensitive and rely on the detection of electrons or ions emitted from the surface under study. These modern probes have been limited to study solid samples. As a result, biological surface analysis in their native liquid environment remains a grand technical challenge.In this work, a high vacuum compatible microfluidic device, namely System for Analysis at the Liquid Vacuum Interface (SALVI). was fabricated, and then used for in situ analysis of biofilms and single mammalian cells by ToF-SIMS in their native liquid environment. Details are described below.1. Fabrication and demonstration of SALVI feasibility in biological sample analysis in the liquid in vacuumA self-contained vacuum compatible microfluidic-based device, System for Analysis at the Liquid-Vacuum Interface (SALVI), was fabricated and applied in biological surface analysis in the liquid environment using ToF-SIMS. The device consists of a poly(dimethylsiloxane) (PDMS) microchannel and a 100 nm thick silicon nitride (SiN) membrane. A small aperture with a diameter of 2 μm on the SiN membrane is dynamically drilled through using ToF-SIMS. The aperture serves as the detection window for in situ detection of liquid surfaces. In addition, results from ToF-SIMS measurement of DI water in SALVI verified the feasibility of SALVI working under high vacuum environment.2. In situ molecular imaging of a hydrated biofilm in SALVI by ToF-SIMS.SALVI was employed for biofilm growth and in situ characterization using ToF-SIMS, which was constructed to enable submicron two-dimensional and three-dimensional chemical imaging of hydrated live biofilms. In this work, we demonstrate the detection of characteristic fatty acid fragments from biofilms and illustrate advantages of hydrated-state ToF-SIMS imaging. We also presented the depth profiling of the biofilm, which showed the punch through of SiN and the detection of biofilm-SiN interface. Three-dimensional imaging of representative fatty acid fragments and principal component analysis (PCA) along the biofilm-SiN interface showed the chemical heterogeneity of biofilm and suggested the possible function of fatty acids in biofilm attachment. This approach provides a novel universal way for directly visualization of spatial and chemical heterogeneity within the complex living biofilm system by dynamic liquid ToF-SIMS.3. In situ single mammalian cell surface analysis in the hydrated state by ToF-SIMS and SIMIn this work we presented the first correlative imaging of single mammalian cells by SIM (structural illumination microscopy) and ToF-SIMS. The former technique was used to confirm cell growth in the channel of SALVI and subsequently guide ToF-SIMS analysis of a single cell. ToF-SIMS depth profiling of a single cell was acquired and was divided into three regions (punch through of SiN membrane, punch through of cell membrane and the detection of cytoplasm). Each region was reconstructed into a 2D image for representative species, which gave a clear visualization of the intensity and distribution changes of these species. Atomic force microscopy (AFM) was used to measure the shape and morphology of the hole. Correlative imaging between SIM and ToF-SIMS enabled the analysis of different locations of a specific cell. Furthermore, both positive and negative ToF-SIMS spectra were presented and showed the characteristic peaks of cell membrane. In addition, cells were treated with zinc oxide nanoparticle (ZnO NP) and analyzed by ToF-SIMS. As a result, Zn+ was detected in cell membrane. PCA distinguished different samples and showed the contributors of these differences and similarities. These first correlative imaging results demonstrated that SALVI could be a promising tool for capturing molecular changes dynamically in a single cell and address the mesoscale challenge underpinning transient molecular interactions leading to complex cell specific changes.4. Selective recognition of the MCF-7 breast cancer cell surface and detection of the cells using aptamer-functionalized magnetic beads and quantum dots based nano-bio-probesA novel strategy for selective collection and detection of breast cancer cells (MCF-7) based on aptamer-cell interaction was developed. Mucin 1 protein (MUC1) aptamer (Apt1) was covalently conjugated to magnetic beads to capture MCF-7 cell through affinity interaction between Apt1 and MUC1 protein that overexpressed on the surface of MCF-7 cells. Meanwhile, a nano-bio-probe was constructed by coupling of nucleolin aptamer AS1411 (Apt2) to CdTe quantum dots (QDs) which were homogeneously coated on the surfaces of monodispersed silica nanoparticles (SiO2 NPs). The nano-bio-probe displayed similar optical and electrochemical performances to free CdTe QDs, and remained high affinity to cells which nucleolin overexpressed through the interaction between AS1411 and nucleolin protein. Photoluminescence (PL) and square-wave voltammetric (SWV) assays were thus introduced to quantitatively detect MCF-7 cells. Improved selectivity was obtained by using both two aptamers as recognition elements simultaneously. Based on the signal amplification of QDs coated silica nanoparticles (QDs/SiO2), the detection sensitivity was enhanced and a detection limit of 85 cells mL-1 was achieved by SWV method. The proposed strategy could be extended to detect other cells, and showed potential applications in cell imaging and drug delivery.