Article pubs.acs.org/cm
Entrapment of Living Bacterial Cells in Low-Concentration Silica Materials Preserves Cell Division and Promoter Regulation Nikolas M. Eleftheriou,†,‡ Xin Ge,†,§ Julia Kolesnik,† Shannon B. Falconer,∥ Robert J. Harris,⊥ Cezar Khursigara,⊥ Eric D. Brown,∥ and John D. Brennan*,† †
Biointerfaces Institute and Department of Chemistry & Chemical Biology, McMaster University, Hamilton, Ontario, Canada L8S 4L8 ‡ Department of Laboratory Medicine, Lund University, SE-22185 Lund, Sweden § Department of Chemical and Environmental Engineering, University of California, Riverside, California 92521, United States ∥ Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada L8S 3Z5 ⊥ Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 S Supporting Information *
ABSTRACT: The entrapment of bacterial cells within inorganic silica materials was reported almost 20 years ago. However, almost all studies to date have shown that these entrapped cells are unable to divide and thus should be expected to have reduced promoter activity. In view of the importance of bacteria as model systems for both fundamental and applied biological studies, it is crucial that immobilized cells retain solutionlike properties, including the ability to divide and display normal promoter activity. Herein we report on a method to immobilize bacterial cells within low-density inorganic silica-based materials, where the cells retain both cell division and promoter activity. Sol−gel processing was used to entrap Escherichia coli cells carrying a variety of green fluorescent protein-linked promoters into sodium silicate-derived materials that were formed in microwell plates. Using a series of assays, we were able to demonstrate that (1) the entrapped cells can divide within the pores of the silica matrix, (2) cellular pathways are regulated in a similar manner in both solution and the sol−gel-derived materials, and (3) promoters in entrapped cells can be specifically induced with small molecules (e.g., antimicrobial compounds) in a concentration-dependent manner to allow assessment of both potency and mode of action. This solid-phase assay system was tested using multiple antimicrobial pathways and should enable the development of solid-phase assays for the discovery of new small molecules that are active against bacteria. KEYWORDS: encapsulation, sol−gel, biosensor, screening, green fluorescent protein, silica, cell entrapment
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INTRODUCTION
small molecules to freely diffuse through the gel, providing a useful platform for cell-based bioreactors and biosensors.1 There are several advantages to using sol−gel-based entrapment as a method to immobilize live cells. Specifically, entrapped cells are viable for up to 1.5 years;16 the process of entrapment does not instigate the expression of major stressresponse genes;3,17 and cells have been shown to remain metabolically active within the gels.16,18 However, these studies and others have been directed toward the development of biosensing platforms19 and bioreactors4 and have probed the response of single promoters rather than more comprehensive evaluations of multiple promoter responses. Furthermore, cell growth within the pores of sol−gel-derived materials has not been reported until recently, using cells entrapped in layered silica thin films.20 Thus, the research completed is far from comprehensive, and it is not known how most cell pathways
The bioapplications of sol−gel-derived biomaterials are broad; proteins, nucleic acids, and a variety of different cells have all been successfully encapsulated into silica or related organic/ inorganic composite matrices.1 In the case of cells, various bacterial,2−4 yeast,5−7 animal,8,9 vegetal,6,10,11 and algal cells12 have been successfully entrapped, although in all cases these cells were unable to divide and thus were physiologically compromised. The success of sol−gel-based entrapment is in large part due to the mild conditions used for preparation of the biocomposites, which generally avoid extreme temperatures and pH and can be performed using aqueous buffers to promote condensation of silica around the cell. In addition, the silica matrix can be readily altered through the use of modified silanes, various additives, and polymers to alter the matrix charge, porosity, and gelation time; material modifications that impart less cellular stress can be determined by feedback from entrapped reporter cells or changes in cell viability.13−15 The resulting mesoporous materials retain the cells while allowing © 2013 American Chemical Society
Received: September 27, 2013 Revised: November 20, 2013 Published: November 26, 2013 4798
dx.doi.org/10.1021/cm403198z | Chem. Mater. 2013, 25, 4798−4805
Chemistry of Materials
Article
Microwell Plate Assays. Promoter expression levels were determined using fluorescence measurements in NUNC and BD Falcon 96-well clear-bottom black plates. The final assay volume in the microwell plate was 300 μL. Generally, gels were prepared within individual wells after mixing of the sol (50 μL), PBS buffer (47 μL), and cell suspension (3 μL); after gelation, M9 minimal medium (200 μL) was added over the top of the hydrogel. Similarly, solution samples were prepared from PBS buffer (97 μL) and cells (3 μL); M9 minimal medium (200 μL) was then added to bring the solution assays to 300 μL. In both sol−gel and solution assays, the cell suspension was present at 1% (v/v) of the total microwell assay volume, providing an identical number of cells in each type of assay. Blank samples without cells had an equal volume of cell-free culture added in lieu of bacterial culture. An Infinite M1000 plate reader and iControl software (TECAN, Männedorf, Switzerland) were used to measure absorbance and fluorescence. Absorbance was collected at 600 nm. Fluorescence parameters were as follows: excitation, 488 nm, bandwidth, 5 nm; emission, 510 nm, bandwidth, 10 nm; bottom read mode. Gain regulation was applied to real-time fluorescence acquisition, unless otherwise noted, to obtain a high fluorescence signal without saturating the photomultiplier tube. Temperature was controlled at 26 ± 1 °C for all measurements, unless otherwise noted. Assays were run in triplicate, and the data points were averaged. Viable Cell Counting. CFU determination was used to indirectly observe cell division over time. Solution and SG samples containing E. coli K12 were prepared in glass vials as described above. Triplicate samples were allowed to incubate at room temperature between 1 and 48 h in 200 μL of M9 minimal medium. After incubation, a series of six 10-fold dilutions into 0.85% (w/v) NaCl was prepared for both solution and SG samples. Before dilution, the SG gels were washed three times with 0.85% (w/v) NaCl to remove any leached cells (typically
