Combined Dielectrophoresis–Raman Setup for the Classification of Pathogens Recovered from the Urinary Tract

Abstract

Rapid and effective methods of pathogen identifications are of major interest in clinical microbiological analysis to administer timely tailored antibiotic therapy. Raman spectroscopy as a label-free, culture-independent optical method is suitable to identify even single bacteria. However, the low bacteria concentration in body fluids makes it difficult to detect their characteristic molecular fingerprint directly in suspension. Therefore, in this study, Raman spectroscopy is combined with dielectrophoresis, which enables the direct translational manipulation of bacteria in suspensions with spatial nonuniform electrical fields so as to perform specific Raman spectroscopic characterization. A quadrupole electrode design is used to capture bacteria directly from fluids in well-defined microsized regions. With live/dead fluorescence viability staining, it is verified, that the bacteria survive this procedure for the relevant range of field strengths. The dielectrophoretic enrichment of bacteria allows for obtaining high quality Raman spectra in dilute suspensions with an integration time of only one second. As proof-of-principle study, the setup was tested with Escherichia coli and Enterococcus faecalis, two bacterial strains that are commonly encountered in urinary tract infections. Furthermore, to verify the potential for dealing with real world samples, pathogens from patients' urine have been analyzed. With the additional help of multivariate statistical analysis, a robust classification model could be built and allowed the classification of those two strains within a few minutes. In contrast, the standard microbiological diagnostics are based on very time-consuming cultivation tests. This setup holds the potential to reduce the crucial parameter diagnosis time by orders of magnitude.

The use of microfluidics and dielectrophoresis for separation, concentration, and identification of bacteria

“…An example of a DEP-Raman involves the use of a quadruple electrode arrangement to concentrate bacteria by negative DEP for Raman analysis [92][93][94].…”

“…Although successful, the design appears impractical as it is meant for small sample volumes (~200 μL) with some of the studies injecting even smaller volumes (10 μL) of concentrated bacteria at the DEP site for successful demonstration [92,93]. Not only is the sample size problematic, but the design is prone to common DEP-related issues of electrode fouling, electrolysis, and Joule heating.…”

“…Along with Raman spectroscopy, other methods generate complex spectra such as Fourier transform infrared spectroscopy (FT-IR) [34,98,99] and surface-enhanced Raman spectroscopy [61,71,94,100]. Due to the complex nature of the spectra, many researchers ease data processing by truncating data using principal component analysis (PCA) prior to classifying bacteria [34,92,[101][102][103][104][105][106].…”

“…For example, several researchers have used a quadruple electrode arrangement to concentrate bacteria by negative DEP for Raman analysis [92][93][94]. Although successful, the design appears impractical as it is meant for small sample volumes (~200 μL) with some of the studies injecting even smaller volumes (10 μL) of concentrated bacteria at the DEP site for successful demonstration [92,93]. Not only is the sample size problematic, but the design is prone to common DEP-related issues as stated previously and is ill-suited to analyze samples containing more than one bacteria at a time.…”

Rapid, culture-independent, optical diagnostics of centrifugally captured bacteria from urine samples

“…22 Raman spectroscopy offers unique benefits in the field of optical diagnostics, 23 as it instantly delivers a complete molecular fingerprint of the investigated specimen without the need for time-consuming and often toxic labeling. 24 This technique has already demonstrated great potential with respect to the identification of bacteria directly from patient samples in previous work by Schr€ oder et al, 25 Kloss et al, 26 and Rusciano et al, 27 although the level of automation is still limited. Therefore, the combination of Raman spectroscopy with microfluidic sample management for the safe handling of real-world patient samples could lead to a major step forward in the field of biomedical diagnostics.…”