Raman and scanning probe microscopy for differentiating surface imprints of E. coli and B. cereus.

Molecularly imprinted polymers (MIPs) are artificial recognition materials mimicking biological recognition entities such as antibodies. The general model of imprinting assumes that functional monomers interact with functional groups present on the target species which leads to cavities complementing the template in surface chemistry and shape thus ensuring recognition. However, to date there is little independent experimental evidence supporting that the surface chemistry in the imprints is tailored to analyte recognition and thus differs from the surface chemistry of the surrounding polymer. Herein, we investigate such chemical differences between imprints of Escherichia coli and Bacillus cereus in poly(styrene-co-DVB) and a commercial acrylate-based polymer by the means of confocal Raman microscopy and PLS-DA. The MIPs were generated using a stamping approach. Peak-force QNM measurements were conducted to rule out residues of bacterial cells in the imprints. While imprints of E. coli and B. cereus could be distinguished based on their Raman spectra in the acrylate-based polymer, differentiation in poly(styrene-co-DVB) was not significant. This could be a result of a higher potential of acrylate functional groups for interacting with lipopolysaccharides and peptidoglycans on bacteria surfaces compared to the phenyl groups of poly(styrene-co-DVB) and emphasizes the importance of the right choice of functional monomers for a specific target analyte.

Investigations on sub-structures within cavities of surface imprinted polymers using AFM and PF-QNM.

Investigations on lithographically formed cavities of surface-imprinted polymers (SIP) can help to gain deeper understanding on cell recognition with SIPs: it is known that surface topography and biomolecules transferred during surface imprinting contribute to cell adhesion. In this work, SIPs synthesized via two different imprinting techniques, namely stamp imprinting and polymerization of Pickering emulsions, were investigated and compared to each other, using atomic force microscopy (AFM) and Peak Force Quantitative Nano Mechanics (PF-QNM). We focused on SIPs based on poly(styrene-co-divinylbenzene) as model polymer and E. coli as model template for cell imprinting. Both imprinting approaches led to cavities that revealed nanostructures within the imprints. Stamp imprinting cavities feature low surface roughness and channel structures that resemble the negative pattern of the bacteria on the stamp and their filaments, while SIPs synthesized via polymerization of Pickering emulsions reveal globular nanostructures accumulating in the imprints. AFM phase imaging and adhesion mapping using PF-QNM show that these globular structures are remainders of the imprinted E. coli cells, most likely lipopolysaccarides, which is not observable in imprints resulting from stamp imprinting.

Raman Studies on Surface-Imprinted Polymers to Distinguish the Polymer Surface, Imprints, and Different Bacteria.

Molecularly imprinted polymers (MIPs) are widely used as robust biomimetic recognition layers in sensing devices targeting a wide variety of analytes including microorganisms such as bacteria. Assessment of imprinting success and selectivity toward the target is of great importance in MIP quality control. We generated Escherichia coli-imprinted poly(styrene-co-DVB) as a model system for bacteria-imprinted polymers via surface imprinting using a glass stamp with covalently immobilized E. coli. Confocal Raman Microscopy was successfully employed to visualize bacteria, imprints, and polymer and to distinguish them from each other. The method has proven highly feasible for assessing if imprinting had been successful. In addition, we developed a method for selectivity investigation of bacteria MIPs based on combining Confocal Raman Microscopy and Partial Least Squares Discriminant Analysis (PLS-DA). The Raman spectra of E. coli and Bacillus cereus were acquired on E. coli-imprinted poly(styrene-co-DVB) and used to establish a PLS-DA model for differentiating between the bacteria species. Model validation demonstrated a correct classification of 95% of Raman spectra, indicating sufficient accuracy of the model for future use in MIP selectivity studies. Simultaneous differentiation of 3 bacteria species (E. coli, B. cereus, and Lactococcus lactis) on E. coli-imprinted poly(styrene-co-DVB) proved more difficult, which might be due to the limited depth resolution of the confocal Raman microscope resulting in the presence of interfering signals from the polymer substrate. It might be possible to overcome this obstacle by selective enhancement of the Raman signals originating from bacteria surfaces, such as tip enhanced Raman spectroscopy.

Biomimetic Sensors to Detect Bioanalytes in Real-Life Samples Using Molecularly Imprinted Polymers: A Review.

Molecularly imprinted polymers (MIPs) come with the promise to be highly versatile, useful artificial receptors for sensing a wide variety of analytes. Despite a very large body of literature on imprinting, the number of papers addressing real-life biological samples and analytes is somewhat limited. Furthermore, the topic of MIP-based sensor design is still, rather, in the research stage and lacks wide-spread commercialization. This review summarizes recent advances of MIP-based sensors targeting biological species. It covers systems that are potentially interesting in medical applications/diagnostics, in detecting illicit substances, environmental analysis, and in the quality control of food. The main emphasis is placed on work that demonstrates application in real-life matrices, including those that are diluted in a reasonable manner. Hence, it does not restrict itself to the transducer type, but focusses on both materials and analytical tasks.

Continuous Flow Reactors from Microfluidic Compartmentalization of Enzymes within Inorganic Microparticles.

Compartmentalization and selective transport of molecular species are key aspects of chemical transformations inside the cell. In an artificial setting, the immobilization of a wide range of enzymes onto surfaces is commonly used for controlling their functionality but such approaches can restrict their efficacy and expose them to degrading environmental conditions, thus reducing their activity. Here, we employ an approach based on droplet microfluidics to generate enzyme-containing microparticles that feature an inorganic silica shell that forms a semipermeable barrier. We show that this porous shell permits selective diffusion of the substrate and product while protecting the enzymes from degradation by proteinases and maintaining their functionality over multiple reaction cycles. We illustrate the power of this approach by synthesizing microparticles that can be employed to detect glucose levels through simultaneous encapsulation of two distinct enzymes that form a controlled reaction cascade. These results demonstrate a robust, accessible, and modular approach for the formation of microparticles containing active but protected enzymes for molecular sensing applications and potential novel diagnostic platforms.

Silica particles with a quercetin-R5 peptide conjugate are taken up into HT-29 cells and translocate into the nucleus.

Intracellular delivery of bioactive polyphenols is currently evaluated as a protective strategy for cells under pharmaceutical stress. To this end, the 20mer R5 peptide from the marine diatom C. fusiformis was N-terminally modified with a quercetin derivative. This polyphenol-peptide conjugate was used to generate homogeneous silica particles under biomimetic conditions that are efficiently taken up by eukaryotic cells without being cytotoxic. However, not only was accumulation in the cytoplasm of living cells observed via electron and fluorescence microscopy but also translocation into the nucleus. The latter was only seen when the quercetin-peptide conjugate was present within the silica particles and provides a novel targeting option for silica particles to nuclei.