RESUMO
Raman spectroscopy provides non-destructive, label-free quantitative studies of chemical compositions at the microscale as used on NASA's Perseverance rover on Mars. Such capabilities come at the cost of high requirements for instrumentation. Here we present a centimeter-scale miniaturization of a Raman spectrometer using cheap non-stabilized laser diodes, densely packed optics, and non-cooled small sensors. The performance is comparable with expensive bulky research-grade Raman systems. It has excellent sensitivity, low power consumption, perfect wavenumber, intensity calibration, and 7 cm-1 resolution within the 400-4000 cm-1 range using a built-in reference. High performance and versatility are demonstrated in use cases including quantification of methanol in beverages, in-vivo Raman measurements of human skin, fermentation monitoring, chemical Raman mapping at sub-micrometer resolution, quantitative SERS mapping of the anti-cancer drug methotrexate and in-vitro bacteria identification. We foresee that the miniaturization will allow realization of super-compact Raman spectrometers for integration in smartphones and medical devices, democratizing Raman technology.
RESUMO
Given the commercial importance of the compounds produced by genetically modified organisms, there is a need for screening methods which facilitate the evaluation of newly developed strains, especially during the phase of proof-of-concept development. We report a time-efficient analysis method for the screening of bacterial strains, which enables the detection of two structurally similar secondary bacterial metabolites. By combining liquid-liquid extraction and surface-enhanced Raman scattering we were able to quantify p-coumaric acid and cinnamic acid, produced by genetically modified E. coli from tyrosine and phenylalanine, respectively. With the simple sample pre-treatment method, and by applying a partial least squares data analysis method, we simultaneously detected the analytes from four E. coli strains cultured in the presence or absence of tyrosine and phenylalanine.
Assuntos
Cinamatos/análise , Escherichia coli/classificação , Escherichia coli/metabolismo , Extração Líquido-Líquido/métodos , Propionatos/análise , Análise Espectral Raman/métodos , Amônia-Liases/metabolismo , Ácidos Cumáricos , Escherichia coli/enzimologia , Fenilalanina/metabolismo , Fenilalanina Amônia-Liase/metabolismo , Tirosina/metabolismoRESUMO
We present the development of an automated centrifugal microfluidic platform with integrated sample pre-treatment (filtration and liquid-liquid extraction) and detection (SERS-based sensing). The platform consists of eight calibration and four assay modules, fabricated with polypropylene using injection molding and bonded with ultrasonic welding. The platform was used for detection of a secondary bacterial metabolite (p-coumaric acid) from bacterial supernatant. The obtained extraction efficiency was comparable to values obtained in batch experiments and the SERS-based sensing showed a good correlation with HPLC analysis.
Assuntos
Escherichia coli/química , Escherichia coli/genética , Extração Líquido-Líquido , Técnicas Analíticas Microfluídicas , Propionatos/análise , Cromatografia Líquida de Alta Pressão , Ácidos Cumáricos , Escherichia coli/metabolismo , Polipropilenos/química , Análise Espectral Raman , Propriedades de SuperfícieRESUMO
During the last few decades, great advances have been reached in high-throughput design and building of genetically engineered microbial strains, leading to a need for fast and reliable screening methods. We developed and optimized a microfluidic supported liquid membrane (SLM) extraction device and combined it with surface enhanced Raman scattering (SERS) sensing for the screening of a biological process, namely for the quantification of a bacterial secondary metabolite, p-coumaric acid (pHCA), produced by Escherichia coli. The microfluidic device proved to be robust and reusable, enabling efficient removal of interfering compounds from the real samples, reaching more than 13-fold up-concentration of the donor at 10 µL min-1 flow rate. With this method, we quantified pHCA directly from the bacterial supernatant, distinguishing between various culture conditions based on the pHCA production yield. The obtained data showed good correlation with HPLC analysis.
Assuntos
Escherichia coli/química , Dispositivos Lab-On-A-Chip , Análise Espectral Raman , Cromatografia Líquida de Alta Pressão , Metabolismo SecundárioRESUMO
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.
Assuntos
Escherichia coli/metabolismo , Propionatos/análise , Ácidos Cumáricos , Escherichia coli/química , Ouro/química , Nanopartículas Metálicas/química , Estrutura Molecular , Propionatos/metabolismo , Análise Espectral Raman , Propriedades de SuperfícieRESUMO
BACKGROUND: Previous studies in Alzheimer's Disease (AD) suggest a benefit from switching from one cholinesterase (ChE) inhibitor to another in the event of treatment failure on the index agent. This observational, open-label study sought to evaluate the efficacy of the ChE inhibitor rivastigmine on cognition, functional autonomy and behavior in patients with mild-to-moderate AD previously treated with other ChE inhibitors (switched patients) as well as in those previously ChE-inhibitor-naive (de novo users). METHODS: Patients were eligible for a switch if they experienced a lack or loss of efficacy or had experienced intolerance to prior ChE inhibitor therapy. Rivastigmine was initiated at a dose of 1.5 mg b.i.d. and titration was done as per standard medical practice. Efficacy was assessed using the mini-mental state examination (MMSE) and an abbreviated version of the Clinician's Global Impression of Change (CGI-C) at Month 3 and Month 6. Caregiver burden was also assessed at Month 6 using a self-rated scale. RESULTS: Overall, 2633 subjects were enrolled in this study. The mean MMSE improved from 20.6 at baseline to 21.5 at Month 6. More patients improved than deteriorated on every domain of the CGI-C. Caregivers felt less burdened after the 6 month evaluation period. Efficacy parameters demonstrated favorable results for both de novo and switched patients, but more so in the first group. LIMITATIONS: Open-label studies have an inherent potential for bias by both the caregiver and the physician. In this study, there was also a large percentage of missing patient records for each of the follow-up visits (Months 3 and 6). CONCLUSIONS: Patients with mild-to-moderate AD switched from previous ChE inhibitor therapy to rivastigmine can obtain measurable benefits, although the treatment effect may be less than in de novo patients. Further research into switching cholinesterase inhibitors is warranted.