3 Results
3.1 QCL-GAP spectra of microorganisms
The vibrational response obtained from the microorganisms using the
QCL-GAP was used to determine the functional groups used for
characterization. Because of the spectral similarities among bacterial
species, it is difficult to discriminate using spectrum profile
differences associated with sample homogeneity at the substrate surface.
Sample distribution in the deposition technique used and the uniformity
of the bacteria on the surface after the sample drying process also
affect the discrimination of bacterial species. These factors can impact
the band characterization and the discrimination processes on bacteria
mixtures. The optical arrangement in the QCL-GAP creates an ellipse that
covers a bigger surface area. This setup makes the beam size
significantly larger than a typical IR system. Analysis of the spectral
profile of the microorganisms under study shows characteristic
signatures that allow identification and discrimination between the
species.
Variation in the sample homogeneity during the deposition in the
substrate is expected during spectral acquisition. However, the QCL-GAP
ellipse effect can overcome those variations by covering more areas to
enhance the signals during the spectrum acquisition. This effect played
a role during the spectrum acquisition, providing more information that
led to acceptable discrimination of the bacteria on pure and mixture of
the bacteria.
The spectral measurements for the substrates (references or blanks) were
measured at multiple regions of the deposited sample in the substrate.
Before measuring the spectra of the neat bacteria and mixtures of
bacteria, the spectrum of the substrate without the analyte is acquired
as a reference. Then, the substrate containing the bacteria was
collected. The instrument allows background subtraction without the need
for additional processing. The performance was also verified before each
run by collecting a blank spectrum using a gold (Au) substrate.
Band assignments in the fingerprint region for the bacteria sample’s
present spectra variations due to the substrate’s reflectivity. In this
experiment, the SS substrates used had a high reflectivity that allowed
acquiring as many signals as possible. It is common knowledge that
higher porosity of the substrate will reduce the signal to noise because
it will absorb the light, resulting in fewer signals. That effect can
lead to losing important information, impacting the resolution, and
complicating the discrimination process.
Representative spectra for the Se and Ml mixture and neatSe and Ml spectra are shown in Figure 2. Although
differences are observed in the mixture profile compared with the neat
bacteria spectra, some bands have similarities. The same effect was
observed for the other mixtures, such as Sa and Ml , shown
in Figure 3, and Sa and Se , shown in Figure 4.