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Monitoring enzymatic degradation of pericellular matrices through SERS stamping.

ABSTRACT: We introduce a surface enhanced Raman spectroscopy (SERS) stamping approach for acquiring cell-surface specific vibrational spectra of individual living cells under physiological conditions. The SERS stamping approach utilizes a nanostructured metal surface on top of a lithographically defined piston that can be translated in 3-dimensions with nanometer resolution to contact living cells in solution with a pristine metal surface. We applied this approach to characterize the chemical composition of the cellular surface of living MCF7 breast cancer cells and to monitor its change upon addition of the enzyme hyaluronidase, which degrades major constituents of the pericellular matrix. Although the cell surface spectra show significant cell-to-cell fluctuations, a statistical barcode analysis of the spectra ensembles reveals systematic changes in the cell surface SERS spectra upon addition of hyaluronidase, which are consistent with a thinning of the pericellular matrix.

SUBMITTER: Yan B

PROVIDER: S-EPMC3461839 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Monitoring enzymatic degradation of pericellular matrices through SERS stamping.

Yan Bo B Hong Yan Y Chen Tianhong T Reinhard Björn M BM

Nanoscale 20120601 13

We introduce a surface enhanced Raman spectroscopy (SERS) stamping approach for acquiring cell-surface specific vibrational spectra of individual living cells under physiological conditions. The SERS stamping approach utilizes a nanostructured metal surface on top of a lithographically defined piston that can be translated in 3-dimensions with nanometer resolution to contact living cells in solution with a pristine metal surface. We applied this approach to characterize the chemical composition ...[more]

PMID: 22659641

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Project description:BackgroundThe recalcitrance of lignocellulosics to enzymatic saccharification has been related to many factors, including the tissue and molecular heterogeneity of the plant particles. The role of tissue heterogeneity generally assessed from plant sections is not easy to study on a large scale. In the present work, dry fractionation of ground maize shoot was performed to obtain particle fractions enriched in a specific tissue. The degradation profiles of the fractions were compared considering physical changes in addition to chemical conversion.ResultsCoarse, medium and fine fractions were produced using a dry process followed by an electrostatic separation. The physical and chemical characteristics of the fractions varied, suggesting enrichment in tissue from leaves, pith or rind. The fractions were subjected to enzymatic hydrolysis in a torus reactor designed for real-time monitoring of the number and size of the particles. Saccharification efficiency was monitored by analyzing the sugar release at different times. The lowest and highest saccharification yields were measured in the coarse and fine fractions, respectively, and these yields paralleled the reduction in the size and number of particles. The behavior of the positively- and negatively-charged particles of medium-size fractions was contrasted. Although the amount of sugar release was similar, the changes in particle size and number differed during enzymatic degradation. The reduction in the number of particles proceeded faster than that of particle size, suggesting that degradable particles were degraded to the point of disappearance with no significant erosion or fragmentation. Considering all fractions, the saccharification yield was positively correlated with the amount of water associated with [5-15 nm] pore size range at 67% moisture content while the reduction in the number of particles was inversely correlated with the amount of lignin.ConclusionReal-time monitoring of sugar release and changes in the number and size of the particles clearly evidenced different degradation patterns for fractions of maize shoot that could be related to tissue heterogeneity in the plant. The biorefinery process could benefit from the addition of a sorting stage to optimise the flow of biomass materials and take better advantage of the heterogeneity of the biomass.

Project description:Bulk tissue stiffness has been correlated with regulation of cellular processes and conversely cells have been shown to remodel their pericellular tissue according to a complex feedback mechanism critical to development, homeostasis, and disease. However, bulk rheological methods mask the dynamics within a heterogeneous fibrous extracellular matrix (ECM) in the region proximal to a cell (pericellular region). Here, we use optical tweezers active microrheology (AMR) to probe the distribution of the complex material response function (?=?'+??, in units of µm/nN) within a type I collagen ECM, a biomaterial commonly used in tissue engineering. We discovered cells both elastically and plastically deformed the pericellular material. ?' is wildly heterogeneous, with 1/?' values spanning three orders of magnitude around a single cell. This was observed in gels having a cell-free 1/?' of approximately 0.5nN/µm. We also found that inhibition of cell contractility instantaneously softens the pericellular space and reduces stiffness heterogeneity, suggesting the system was strain hardened and not only plastically remodeled. The remaining regions of high stiffness suggest cellular remodeling of the surrounding matrix. To test this hypothesis, cells were incubated within the type I collagen gel for 24-h in a media containing a broad-spectrum matrix metalloproteinase (MMP) inhibitor. While pericellular material maintained stiffness asymmetry, stiffness magnitudes were reduced. Dual inhibition demonstrates that the combination of MMP activity and contractility is necessary to establish the pericellular stiffness landscape. This heterogeneity in stiffness suggests the distribution of pericellular stiffness, and not bulk stiffness alone, must be considered in the study of cell-ECM interactions and design of complex biomaterial scaffolds. STATEMENT OF SIGNIFICANCE:Collagen is a fibrous extracellular matrix (ECM) protein widely used to study cell-ECM interactions. Stiffness of ECM has been shown to instruct cells, which can in turn modify their ECM, as has been shown in the study of cancer and regenerative medicine. Here we measure the stiffness of the collagen microenvironment surrounding cells and quantitatively measure the dependence of pericellular stiffness on MMP activity and cytoskeletal contractility. Competent cell-mediated stiffening results in a wildly heterogeneous micromechanical topography, with values spanning orders of magnitude around a single cell. We speculate studies must consider this notable heterogeneity generated by cells when testing theories regarding the role of ECM mechanics in health and disease.

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Monitoring enzymatic degradation of pericellular matrices through SERS stamping.

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Monitoring enzymatic degradation of pericellular matrices through SERS stamping.

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