Boisen, Anja

Burger, Robert

Jendresen, Christian Bille

Morelli, Lidia

Nielsen, Alex Toftgaard

Rindzevicius, Tomas

A standard protocol for analysis of microbial factories requires the screening of several populations in order to find the best performing ones. This is done with standard analytical methods (e.g. HPLC) with an expensive and time-consuming process. Surface Enhanced Raman Spectroscopy (SERS) is a highly sensitive spectroscopic technique which only requires drying a small volume of solution on an active substrate, with an analysis time of few minutes. Here we demonstrate the use of SERS to discriminate between two different bacterial populations based on detection of p-coumaric acid (pHCA) in cell supernatant. pHCA is a valuable secondary metabolite of genetically modified E. coli[1]. It is produced through deamination of tyrosine, and it has strong Raman and SERS activity[2],[3]. Gold capped silicon nanopillars were used as sensing substrates[4]. At first, they were successfully used to detect pHCA spiked in culture medium, in the same concentration range (10-4 – 10-5 M) commonly found in cell supernatant. For supernatant analysis, triplicate cultures of FjTAL modified (P strains) and control (C strains) E.coli strains were carried out according to the methods described by[5] and shown in Fig.1. Samples of cell supernatant were extracted from each culture at 0, 3, 24 and 48 h post seeding and their pHCA content was measured with HPLC[5]. For SERS analysis, aliquots of supernatant were diluted 10-fold with MilliQ water, and 1 μL droplets were dried on the SERS substrates. A MatLab analysis was performed to extract the height of the significant peak at 1169cm-1, with the results shown in Fig.2. The amplitude of the peak shows a different trend for P and C strains. A similar trend is obtained from HPLC. These promising results open up new possibilities for the use of SERS for high-throughput and automated evaluation of bacterial factories, allowing parallel analysis and discrimination of different strains

Online Research Database In Technology

Surface Enhanced Raman Spectroscopy detection of p-coumaric acid from cell supernatant using gold-capped silicon nanopillar substrates

The purpose of the project is to use Surface Enhanced Raman Spectroscopy (SERS) to discriminate between two different bacterialpopulations, based on their p-coumaric acid (pHCA) production. The pHCA concentration is measured in a droplet of diluted supernatantdried on SERS substrates, using a Raman microscope. By analyzing the SERS signal of pHCA from the supernatant, considering the peakheight at the characteristic frequency (1169 cm-1) it is possible to distinguish between a producing and control strain, as also confirmedby HPLC analysis

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Downloaded from orbit.dtu.dk on: Dec 18, 2017Surface Enhanced Raman Spectroscopy detection of p-coumaric acid from cellsupernatant using gold-capped silicon nanopillar substratesMorelli, Lidia; Jendresen, Christian Bille; Burger, Robert; Rindzevicius, Tomas; Nielsen, Alex Toftgaard;Boisen, AnjaPublication date:2016Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Morelli, L., Jendresen, C. B., Burger, R., Rindzevicius, T., Nielsen, A. T., & Boisen, A. (2016). Surface EnhancedRaman Spectroscopy detection of p-coumaric acid from cell supernatant using gold-capped silicon nanopillarsubstrates. Poster session presented at Biosensors 2016, Gothenburg, Sweden.SURFACE ENHANCED RAMAN SPECTROSCOPY DETECTION OF p-COUMARIC ACID FROM CELL SUPERNATANT USING GOLD-CAPPED SILICON NANOPILLAR SUBSTRATES  L. Morelli1, C. Bille Jendresen2, K. Zor1, T. Rindzevicius1, M. Stenbæk Schmidt1, A. Toftgaard Nielsen2,  A. Boisen1   1 Department of Micro- and Nanotechnology, Technical University of Denmark, Kgs. Lyngby, DENMARK 2 The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, DENMARK  The purpose of the project is to use Surface Enhanced Raman Spectroscopy (SERS) to discriminate between two different bacterial populations, based on their p-coumaric acid (pHCA) production.  The pHCA concentration is measured in a droplet of diluted supernatant dried on SERS substrates, using a Raman microscope. By analyzing the SERS signal of pHCA from the supernatant, considering the peak height at the characteristic frequency (1169 cm-1) it is possible to distinguish between a producing and control strain, as also confirmed by HPLC analysis. SERS: fabrication and working principle Fig.1: Process steps for Au capped nanopillars (NPs) fabrication (courtesy of Kaiyu Wu) [1]: (a) silicon NPs are fabricated by maskless reactive ion etching (RIE); (b) O2 cleaning removes the Si RIE contaminants from the Si surface; (c) a Au metal film is deposited by e-beam evaporation. (d) When a droplet of solution is dried on the substrate, the surface tension tends to pull the Au NPs together, forming irreversible clusters and trapping the analyte molecules between the NPs. Furthermore, E-field hotspots are created when two NPs lean close to each other. (e) SEM picture of leaning pillars. Bacterial cultures and measurements Outlook and conclusions In this work we demonstrated that SERS is a rapid and effective tool for qualitative screening of bacterial strains, based on the amount of synthetized secondary metabolites (e.g. pHCA). These results open up new possibilities for high-throughput quantitative analysis. Currently we are focusing on improving sensitivity by extracting pHCA in organic solvent and on integration of the assays on automated and high-throughput microfluidic platforms, such as lab-on-a-discs. [1] K. Wu, T. Rindzevicius, M. S. Schmidt, K. B. Mogensen, A. Hakonen, and A. Boisen, J. Phys. Chem. C, vol. 119, no. 4, pp. 2053–2062, 2015.  [2] C. B. Jendresen, S. G. Stahlhut, M. Li et al., Appl. Environ. Microbiol., vol. 81, no. 13, pp. AEM.00405–15, 2015. Lidia Morelli Technical University of Denmark, Department of Micro and Nanotechnology  phone: +45 91 73 43 40,   email: lmor@nanotech.dtu.dk  a) b) c) d) Salt dilution References Aim of the Project e) 200 nm Validation with HPLC a) b) c) d) e) f) Incubation at 30 °C, 250 rpm Centrifugation and sterile filtration 1 µL of 10-fold diluted supernatant on the substrate Droplets drying and pillars leaning SERS acquisition Fig.2: Bacterial culture, supernatant extraction and SERS evaluation: (a) and (b) genetically modified E.coli are cultured in triplicates [2]; (c) aliquots of bacterial solution are extracted at 0, 3, 24 and 48 h, centrifuged and sterile filtered to extract cell supernatant; (d) and (e) 1 µL of supernatant diluted 10-fold with water is dried on SERS substrates. The SEM pictures show that the pillars stand vertically before wetting, and they lean towards each other after drying. (f) 5x5 maps are acquired on the droplet area with a DXR Raman microscope (Thermo Fisher Scientific Inc.) at 780 nm, 1 mW, 10x objective, 25 µm slit. Fig.4: (a) Concentration of pHCA in cell supernatant for producing (P, black) and control (C, red) strains measured with  HPLC. Each point is the average of 3 measurements, each one obtained  from one strain. (b) SERS signal at 1169 cm-1 after baseline correction. Each point in the graph is the average of 3 maps of 25 points, whereas the error bars represent the standard error of the mean, calculated on the 3 average values. Fig.3: 100 µM pHCA spiked in culture medium diluted with MilliQ water in different ratios (1:2, 1:5, 1:10, 1:20). The signal increases with medium dilution, as higher salt concentration clogs up the active surface and decreases the signal. 1:10 dilution was chosen as a compromise between signal intensity and dilution for measurements in the supernatant. b) a) Inoculation of GM E.coli in M9 medium 

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Surface Enhanced Raman Spectroscopy detection of p-coumaric acid from cell supernatant using gold-capped silicon nanopillar substrates

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