Surface Enhanced Raman Scattering for Quantification of p-Coumaric Acid Produced by Escherichia coli

Abstract

The number of newly developed genetic variants of microbial cell factories for production of biochemicals has been rapidly growing in recent years, leading to an increased need for new screening techniques. We developed a method based on surface-enhanced Raman scattering (SERS) coupled with liquid-liquid extraction (LLE) for quantification of p-coumaric acid (pHCA) in the supernatant of genetically engineered Escherichia coli (E. coli) cultures. pHCA was measured in a dynamic range from 1 μM up to 50 μM on highly uniform SERS substrates based on leaning gold-capped nanopillars, which showed an in-wafer signal variation of only 11.7%. LLE using dichloromethane as organic phase was combined with the detection in order to increase selectivity and sensitivity by decreasing the effect of interfering compounds from the analytes of interest. The difference in pHCA production yield between three genetically engineered E. coli strains was successfully evaluated using SERS and confirmed with high-performance liquid chromatography. As this novel approach has potential to be automated and parallelized, it can be considered for high-throughput screening in metabolic engineering.

Optimizing silver‐capped silicon nanopillars to simultaneously realize macroscopic, practical‐level SERS signal reproducibility and high enhancement at low costs

“…Based on the results above, it is reasonable to set p = 18 mTorr as the optimized RIE chamber pressure for obtaining high‐density Si NPs, as applying it produces NPs of the highest density (∼48 NPs μm −2 ) with a uniform height. It should be pointed out that in previous reports related to the NPs made by a lithography‐free process, only substrates with NPs of low densities less than ∼19 NPs μm −2 were used.…”

Quantification of a bacterial secondary metabolite by SERS combined with SLM extraction for bioprocess monitoring

“…Donor samples were acidified at pH 1 with H 2 SO 4 0.5 M (10% v/v) , whereas 10 mM phosphate buffer (PB), pH 7.4, was used as acceptor buffer for SLM extraction. For pHCA production assays the E. coli cells were grown in minimal M9 medium, 27 containing 10 g/L glucose, 2 mM Tyr, 1 mM IPTG and Wolfe's Vitamin solution purchased from ATCC® (LGC Standards, UK) as extensively described by Morelli et al 23 Aqueous solutions were made with ultrapure water obtained from a Milli-Q purification system (Millipore Corporation, Billerica, MA, U.S.). All the chemicals were purchased from Sigma-Aldrich, unless otherwise stated.…”

“…coli control and pHCA producing strains were constructed as described by Jendresen et al, 7 from the expression strain BL21(DE3)pLysS (Invitrogen/Life Technologies), carrying the extrachromosomal cloning vector pCDFDuet-1 or a derived plasmid encoding the tyrosine ammonia-lyase FjTAL. The pHCA producing strain was grown with 2 mM Tyr in different medium compositions (complete M9 medium, 23 absence of IPTG or absence of vitamins). The control strain, characterized by the absence of FjTAL, was grown without Tyr and IPTG in M9 medium.…”

“…All the spectra were collected 3 times for 0.05 s. 3 maps of 48 spectra each were collected on the surface of each chip, with a 100 µm collection step. The peak height at 1169 cm -1 was used for quantification, according to the methods previously described by Morelli et al 23 The average Raman intensity and the corresponding standard deviation for each sample were calculated over the average values of the maps collected on the same chip.…”

“…There are numerous studies focused on the development of sample preparation and extraction techniques in combination with SERS detection, especially when dealing with complex sample matrices. 22,23 In our previous work, 23 we demonstrated the advantages of combining traditional LLE with SERS detection and successfully screened different E. coli strains based on the produced pHCA quantity. However, LLE proved to have some disadvantages, since it required several manual sample handling steps and the usage of toxic solvents.…”