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Capillary Electrophoresis Absorption Detector

#G-1979


An Improved Capillary Electrophoresis Absorption Detector for Studying Bacteria Reactions

Tech Area / Field

  • CHE-ANL/Analytical Chemistry/Chemistry
  • BIO-MIB/Microbiology/Biotechnology
  • ENV-MIN/Monitoring and Instrumentation/Environment
  • INS-MEA/Measuring Instruments/Instrumentation

Status
3 Approved without Funding

Registration date
26.09.2011

Leading Institute
Tbilisi State University, Georgia, Tbilisi

Collaborators

  • Lawrence Berkeley National Laboratory, USA, CA, Berkeley

Project summary

The goal of the proposed project is to design, build, and test a new capillary electrophoresis (CE) absorption detector. Initially, we will develop a highly sensitive detector for studying bacteria-metal interaction using capillary electrophoresis and atom-absorption spectrometry. The new detector technology can also be used with other existing analytical methods. We will extend this capability to other challenging biological systems in the future, such as bacteria-host cells interactions.

We expect our proposed technology to increase the sensitivity (at least ten folds) of the CE method and also enhance the accuracy for studying cellular reactions with chemicals or biological materials or stressors. In this proposed project, we will use bacteria-metal interactions as our first test bed to confirm our technology.

Capillary electrophoresis (CE) has found increasing applications in solving a range of important analytical problems, including those in pharmaceutics and in modern biological and environmental sciences. The reason for such a widespread application of CE is its high responsiveness (speed) and its high efficiency in separating the ionic species in solution under the influence of an electric field.

The CE technology has been developed intensively over the last decade because of its promise as a tool with high analytical performance. In CE devices, detectors are based on either the direct absorption of light by the species in the samples , or the fluorescence of the species after absorbing the transmitted light. Due to the universal character of the light absorption in the ultraviolet (UV) region, this method is widely popular and is used almost in every commercially available CE devices. The project purpose is studying bacteria-metal interaction with capillary electrophoresis, atom-absorption spectrometry and other methods

Motivation, challenges, and strategy.

CE devices based on absorption detectors are known for their high sensitivity; however for some important biological problems, such as bacteria or other cellular reactions to their immediate environments, the current sensitivity is insufficient. Our experience show that the current performance of absorption detectors have not reached their maximum potential because of several factors: 1) the design of measurement scheme of absorption detectors is not optimized for small samples with dilute chemical concentrations; 2) the volume of specimen under study is very small because of the nature of the study; 3) the absorption of light (detection) occurs wthin an absorption area, wich is small (thin) due to the small diameter of the capillary, and the light intensity available for detection within an absorption area is small. In order to increase the CE absorption performance, we propose to improve the sensitivity of detectors, to increase the size of the absorption area, and to employ newly developed Z-shaped capillaries. The following summarizes the philosophy underneath our proposed strategy which we believe will enable us to overcome the challenges by effectively implementing the above three key elements in our design..

In many CE devices, a monochromatic light with a constant intensity is incident on the capillary (and thereby dictating the absorbing area). Then the electric signal from the photodetector is fed to a direct current (DC) amplifier and then to a recorder. A key drawback of such systems is that, the photodetector may absorb parasitic (spurious) light which can distort the output signal. Furthermore, the output signal is also distorted by noises from the system. These interfering signals have low frequencies. Since the recording system has no barrier for low frequency noise, these signals interfere with the detection limit of the method.

In order to eliminate the interference, we propose the following. First, we will modulate the intensity of the light and connect the photodetector to a narrow-band amplifier. Such a recording system transmits only (just) the noise, frequency of which coincides both with the frequency of the modulation of light intensity of light source and with the band-pass of the narrow-band amplifier. The system performance will be improved further (a system may have even better characteristics) by connecting the narrow-band amplifier to a synchronous detector (see block diagram on p.16); therefore the system under test is fed by a controlling (stimulus) signal with a specific modulation frequency from the modulator.

In the system described above, the quality of the output signal is continuously affected in at least two ways, Firstly, the signal quality can be impaired by the instabilities (or drifts) of the intensity of the light, usually occur in the low frequency region, Our experience shows that this effect is especially pronounced when the system is operated at the high-sensitivity modes of operation, despite the measures taken to stabilize the intensity of the light. It prevents one to detect absorption signals, wich are commensurable to or less than signals, resulted from the instabilities in the intensity of light, that limits the detection limit. Secondly, the quality of the output signals can also be impaired by the instabilities in the coefficients of amplification of the photodetector and the amplifier. The signal instabilities, resulted from the instabilities in the coefficients of amplification of the photodetector and the amplifier, prevents one to detect absorption signals reliably, which also impairs the detection limits. It is known that the coefficient of amplification changes because of the instability of the applied voltage and/or the changing temperature. This often leads to a increase in the detectable light intensity. This causes uncertainties, and makes worsens the accuracy and the detection limit; (to a decrease in the accuracy and to a deteriorate, impair, the detection limit) especially when it is necessary crucial to evaluate the concentration of chemical fractions in a very small sample.

Our proposed CE absorption device will enable one to minimize the above system deficiencies significantly, and to improve the detection limit (sensitivity) and accuracy.

Approach

Details of the methods and approach are in Section 6 (Technological Methods and Approach). In our proposed device, we will develop, and test new detector circuits, that are specifically designed to overcome these two key system challenges. Several new designs of absorption detectors with different optical and signal registration systems will be developed and constructed, and their analytical characteristics will be studied. Together, we can compensate the system noise (described above) and accurately detect and identify the fractional species in the sample solution at dilute concentrations.

Model biological system for CE detector development

We plan to use bacteria-chromium interactions to test the performance of our CE technology development.

Bacteria are a good test bed for the technology development for the following reasons: Firstly, their surface contains a variety of charge, which depends on the bacteria semblance and its physiological condition, and secondly bacteria are more easily managed (relative to mammalian cells). In our laboratory, CE method has been successfully used to characterizing the surface properties of different bacterial strains, including those isolated from a USA site polluted with mixtures of radionuclide, heavy metals, and organic solvents. Our previous experience show that if bacteria are subjected to minerals or metal ions, their surface charge and their mobility will change too. In our model biological system, the electrophoretic mobility of bacteria will be used to characterize the changes in bacteria caused by metal-induced stress. During electrophoresis the experimental conditions (buffer pH and composition, the capillary column, temperature, applied voltage) are invariant. Then the characteristic electrophoretic mobility (EM) of bacteria can be related to changes induced by metal agents.

At present we have several strains of Cr(VI) reducer Arthrobacter spp. which are isolated from basalt samples of the most polluted regions in Georgia. We’ll use these bacteria as our model organismsstrains (will be investigated). Since it is also possible to separate metal ions by means of the CE method. We will test the performance of our new detection instrument to separate bacteria and metal ions in the mixture, to characterize their surface properties, and to determine how metal-induced stress would alter the bacterial surface properties.

Chromium and co-contaminants

Our previous work (see reference list) show that some Cr(VI)-resistant bacteria absorbs the ions of Cr (VI) with a great intensity. Cr(VI) transforms into the trivalent form and it happens into the bacteria with the scheme: Cr(VI) → Cr(V) → Cr(IV) → Cr(III) and Cr(III) accumulates. The studying of the mentioned processes has a great scientific cognitive (instructional) importance implications in bioremediation of Cr in environment and their impacts on ecological receptors. In addition (except these), it may have a big practical use in environmental bioremediation. With the presence of different metals as co-contaminants, which occur commonly, new biochemical processes might occur, which might lead to either intensification or retardation of the processes induced by a single metal species.

The proposed detection system is expected to have sufficient sensibility not only to detect effect of chromium of different species on bacteria, it also can be used to detect effect in the presence of other metal ions as co-contraminants It’s possible to observe the mentioned processes by studying the molecule absorption in bacteria. Studying and comparing the absorption of bacteria and Cr compounds, it’s possible to get an important informacion about insight into the processes of metal ion transformation and about the common detoxification processes occuring in bacteria.

Experimentals

Many members (see Section 8) of this project have been taking part in previous studies bacteria’s electrophoresis mobility. They introduced heavy metal-containing solutions of different concentrations to bacteria populations over a range of exposure time. In this proposed work, similar experiments will be performed, but we will use the improved CE absorption detector method to characterize the systems. A simple gold standard method of increasing the sensitivity (five folds) is to include an additional chemical treatment of the inner surface of a quartz capillary in the detection area, thereby increasing the absorption area. We will then compare results from the two different CE methods.

Our expertise and knowledge about CE instrumentation design and performance, and our biochemical understanding of this microbial system will provide the foundation for this project to be realized successfully.


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