Spectroscopy Analysis: Illuminating Our Synthetic Biology Research
Discover how the Hydro Guardian utilizes spectroscopy to analyze and understand our samples. Explore the techniques and findings that have been pivotal in improving our project.
Discover how the Hydro Guardian utilizes spectroscopy to analyze and understand our samples. Explore the techniques and findings that have been pivotal in improving our project.
In the following section, we describe in detail the spectroscopic investigation that we conducted to improve the DESIGN and LEARN background information that is useful for the successful completion of the Engineering Cycle of our whole project. In conjunction with our Model and Interviews of stakeholders, we aimed to LEARN about limits, challenges and demands for the biosensor concept.
However, we have also already kept the engineering cycle in mind during these preliminary physics-based experiments and have applied and cycled all of its steps to improve these spectroscopy experiments.
There are many methods for detecting antibiotics and heavy metals in water with different detection limits, advantages, and disadvantages. To verify our final HydroGuardian biosensor, we initially chose to use Raman spectroscopy.
Raman spectroscopy is a method based on Raman scattering. This effect occurs when light gets scattered on a molecule and some of its energy is being transferred onto the molecule. Having less energy than before now, the frequency of the light changes. This frequency shift is specific for certain molecules, which can be used to identify unknown molecules in a sample. Text
The goal was to determine the limit of detection for Raman spectroscopy and test state of the art physical measurement methods.
The Raman spectroscopy setup consists of a laser at 532 nm wavelength, microscope slides for the samples, a stage, and a spectrometer for detecting the signal that can be analyzed with the Enlighten software. The laser illuminates the microscope slide with the sample and the frequency shift is detected by the spectrometer.
Using the described setup, different spectra were recorded for carbenicillin, ampicillin and copper sulfate in different concentrations. For better results, some samples were left to dry on the microscope slide. The resulting spectra can be seen in the figures below.
The method worked well for highly concentrated copper sulfate that has been left to vaporize, showing clearly visible peaks. However, it did not work as well with the antibiotics. This was due to the fluorescence signal of the sample interfering with the spectrum.
For further experiments, quartz glass should be used instead of a microscope slide. This could minimize the fluorescent effects. Additionally, an objective should be used to increase the amount of light collected by the setup. These changes could improve the metal spectra and lead to more consistent records when repeating measurements. For better antibiotic spectra, literature suggests using surface enhanced Raman spectroscopy (SERS) instead. With SERS, a silver or gold nano structure is introduced to the former set up. The sample should be placed on the metal, resulting in an improved spectrum due to the enhancement of the signal. Text
However, our team did not have the opportunity to use SERS. Instead, we continued the measurements with FTIR.
In the second cycle of our project, we focused on utilizing Fourier-transform infrared spectroscopy (FTIR) to detect residues of antibiotics and heavy metals. FTIR allows the identification of chemical bonds based on their characteristic spectral fingerprint peaks obtained through infrared absorption measurements. This method was explored as a viable alternative to Raman spectroscopy.
Our design phase was centered around the following key points: We aimed to establish the detection limits for antibiotic residues and heavy metal ions in water using FTIR spectroscopy. We reviewed the current - state of the art - physical measurement techniques to understand the capabilities and limitations of existing methods.
We aimed to gain information about the complexes by mixing ampicillin and heavy metal solutions and analyzing the final spectrum. If complexes are formed instead of a mere mixture, then the spectrum would not only show the peaks of the heavy metal together with those of the antibiotic, but new peaks would form, or the existing ones would shift due to the foundation of new bonds.
We have access to a FTIR spectrometer (PerkinElmer FT-IR Spectrometer Spectrum Two). The device operates by passing infrared light through a sample, where different molecules absorb light at specific wavelengths, creating a spectrum that acts as a molecular fingerprint.
To validate our design, we conducted a series of tests using a serial dilution method. We tested various concentrations of ampicillin and copper sulfate in water.
Vaporized stains of 1 µL each were measured, with three samples per concentration. We also combined antibiotic and heavy metal solutions of small concentrations to evaluate their mixed spectra and complex formation behavior.
From our testing phase, we derived several insights:
We were able to identify absorption peaks according to literature Text, in the region of interest (500 – 1800 1/cm), that are indicative of the presence of specific bonds representing the ampicillin and copper sulfate.
The spectra of our measurements are showing some of the same peaks as in literature for ampicillin and the complex Text (see red marked ones). The mixed solution of ampicillin (10 mM) and copper sulfate (10 mM) is partially showing peaks of both single spectra. Nevertheless, it is not possible to evaluate if it has become a complex. Further statements would require further analysis of the samples and adding other investigation techniques.
Our FTIR measurements revealed a detection limit of approximately 1 mM for ampicillin and 0.1 mM for copper sulfate, respectively. At this concentration, the minimum detectable amount could still be identified through its characteristic spectral fingerprint peaks.
The detection limit measured with the FTIR is still relatively high compared to the limit of detection which is stated by the Umwelt Bundesamt to be 0.2 ng/L for ampicillin in Germany Text. This is due to the lower measurement accuracy attributed to the wavelength and absorption properties of water, which is indicating a need for further refinement and sensitivity enhancement.
Overall, our use of FTIR in detecting water contaminants demonstrated both its potential and its current limitations. The next step will focus on improving the detection sensitivity to meet environmental monotoring standards more effectively.
| AMP [1/cm] Text | Cu-AMP compl. [1/cm] Text | AMP [1/cm] | CuSO_4 [1/cm] | Compl [1/cm] |
|---|---|---|---|---|
| 600 | 600 | 599 | 599 | 613 |
| 1025 | 1022 | 699 | 862 | 699 |
| 1455 | 1466 | 788 | 1054 | 979 |
| 1607 | 1625 | 1129 | 1640 | 1054 |
| 1733 | 1670 | 1392 | 1129 | |
| 1456 | 1370 | |||
| 1520 | 1459 | |||
| 1594 | 1527 | |||
| 1626 |
In the 3rd cycle, we are exploring the potential of Whispering Gallery Mode Resonators (WGMR) for advanced sensing applications. WGMR represent a relatively new technique with great potential in the field of biosensing and environmental monitoring. The principle of WGM is based on a standing wave that propagates along a concave surface, enabling highly sensitive detection capabilities.
The sensing mechanism of WGMR relies on detecting mode shifts within the resonator cavity. These shifts occur due to changes in the local refractive index near the resonator's surface. When a particle comes close to the resonator, it causes an increase in the local refractive index. This change is captured by the evanescent field of the light wave, which travels along the surface of the resonator through total internal reflection. The interaction between the evanescent field and the altered refractive index leads to a mode shift, which can be measured to detect the presence and properties of the particle.
With the WGMR, we aim to improve the sensitivity and precision of the limit of detection measurements. The resonator should be available for everyone, low cost and easy for doing it yourself.
A setup with a tunable 780 nm laser was designed and realized (see setup picture). The WGMR consists of a dish to which several glass beads (r = 1 mm) have been glued using a UV-curable glue. The laser beams is guided by several mirrors through a 50/50 beamsplitter and an 4x objective (NA = 0.2) onto the dish’s bottom with an angle of 45°. The light is coupled into the bottom, propagating inside of it by total internal reflection, which also couples into the glass beads by the evanescent field on the dish’s surface. The dish is placed in a self-designed and 3d-printed mounting. The light of the glass beads should be detected via camera, which is not yet integrated in the setup. A part of the light is reflected through the beamsplitter leading to the photodiode which detects the photocurrent.
The light was successfully coupled into the setup. An attempt was made to couple the light into the resonator and to find the resonance of the glass spheres by tuning the laser wavelength. The laser was tuned in the range from 1 V to 63 V. At the same time, the light from the glass beads was recorded using a Logitech camera. No change in light intensity could be detected with the naked eye. Another attempt was made with distilled water, but again there was no change.
WGMRs have great potential as they enable very sensitive detection. However, the way the setup was realized by us, an identification of the particles in the water is not possible. Only a contamination of the water can be measured if resonance is set with pure water. The glass beads would glow when they are in resonance with pure water and no longer glow as soon as a particle that is not a water molecule and has a higher refractive index, is close to the beads, which would lead to a mode shift.
The next step would be to implement a remote camera. The images would then be evaluated with a image processing programme, so that the smallest changes in intensity can be registered.
