X-Ray Fluorescence in Biological Sciences. Группа авторов

systems is very important. The techniques which can normally be used for such determinations are ICP‐MS, ICP‐OES, atomic absorption spectrometry with either flame (FAAS) or electro thermal atomization (ETAAS), etc. In order to avoid severe interferences, these techniques generally require the digestion of the sample and matrix separation. In addition, the matrix removal makes the solution used for analysis less viscous and free flowing during atomization for ICP‐OES and ICP‐MS analysis. Further, the sample requirement for ICP‐OES and ICP‐MS is a few ml of solution, which requires at least a few mg of biological sample. These samples can also be analyzed by TXRF with almost similar detection limits as by other techniques. However, TXRF has some advantages over these techniques, e.g. the sample requirement in TXRF is very small, all metals and non‐metals, from C to U, can be analyzed with different detection limits and a simple quantification procedure [11]. As stated above, the matrix removal is necessary in TXRF analysis also ‐ not for avoiding interference but to make TXRF conditions feasible and reduce background. Since matrix elements present in biological samples are mostly low Z elements, the matrix shall not pose an appreciable absorption enhancement effect, but rather scatter the X‐rays intensively leading to increased background or poor detection limits. After some modifications in sample preparation, it is possible that these samples can be directly analyzed by TXRF. Thus, due to these advantageous features, such as a sample requirement of microgram or lesser quantities, the possibility of multi‐element analysis from C to U alike for metals and nonmetals, direct elemental analysis, as well as simple quantification, TXRF analysis is well suited for biological samples.

      In addition to trace element determinations, the oxidation state determination of the elements present in a biological system, or elemental speciation is also very important. Only a few techniques are available for speciation studies, especially at trace levels of elemental concentration and using very small amounts of sample. TXRF based X‐ray absorption near edge spectroscopy (TXRF‐XANES) can be used for such studies. However, such studies can be performed using Synchrotron light sources. For such applications, the X‐ray beams of variable energies, covering the absorption edge of the element being studied, are allowed to fall on the sample at an angle less than the critical angle and the TXRF spectra are measured for each energy for a short duration by varying the excitation energy in steps. The TXRF emission spectra are converted to XANES spectra and the oxidation state of the analyte element is determined using a similar spectra of standards. This kind of study is very useful and can be done using Synchrotron light sources, but is not possible with conventional lab based TXRF spectrometers, especially at such trace levels and with very small amounts of sample [8].

      The attractive features of TXRF for biological samples can be summarized as given below:

      1 Requirement of very small sample amounts e.g. a few microlitres or micrograms.

      2 Non‐destruction / non consumption of the sample during analysis.

      3 Multi‐elemental determinations of metals and nonmetals alike in the same sample.

      4 Detection limits comparable to other trace element determination techniques, e.g. ICP‐OES and ICP MS.

      5 Simple quantification and sample preparation.

      6 Possibility of direct sample analysis.

      7 Elemental Speciation in TXRF‐XANES mode.

References

      1 1 Carvalho, M.L., Magalhães, T., Becker, M., and Bohlen, A.V. (2007). Trace elements in human cancerous and healthy tissues: a comparative study by EDXRF, TXRF, synchrotron radiation and PIXE. Spectrochim. Acta Part B At. Spectrosc. 62: 1004–1011.

      2 2 Mulware, S.J. (2013). Trace elements and carcinogenicity: a subject in review. Biotechniques 3: 85–96.

      3 3 Pemmer, B., Roschger, A., Wastl, A. et al. (2013). Spatial distribution of the trace elements zinc, strontium and lead in human bone tissue. Bone 57 (1): 184–193. https://doi.org/10.1016/j.bone.2013.07.038.

      4 4 Uo, M., Wada, T., and Sugiyama, T. (2015). Applications of X‐ray fluorescence analysis (XRF) to dental and medical specimens. Jpn. Dental Sci. Rev. 51: 2–9.

      5 5 Van Grieken, R.E. and Markowicz, A. (1993). Handbook of X‐Ray Spectrometry, 2e. Marcel Dekker Inc.: New York.

      6 6 Bertin, E.A. (1984). Principle and Practice of X‐Ray Spectrometric Analysis, 2e. Plenum Press: New York.

      7 7 Misra, N.L. (2014). Advanced X‐ray spectrometric techniques for characterization of nuclear materials: an overview of recent laboratory activities. Spectrochim. Acta Part B 101: 134–139.

      8 8 Misra, N.L. (2014). Characterization of nuclear materials by total reflection X‐ray fluorescence spectrometry. J. Radioanal. Nucl. Chem. 300: 137–145.

      9 9 Yoneda, H. and Huriuchi, T. (1971). Optical flats for use in X‐ray spectrochemical microanalysis. Rev. Sci. Instrum. 42: 1069.

      10 10 Klockenkamper, R. (1996). Total Reflection X‐Ray Fluorescence, Analysis, Chemical Analysis, vol. 140. New York: Wiley.

      11 11 Kunimura, S. and Kawai, J. (2007). Portable total reflection X‐ray fluorescence spectrometer for nanogram Cr detection limit. Anal. Chem. 79: 2593–2595.

      Note

      1 ^* Retired, Email: [email protected]

5 Micro X‐Ray Fluorescence and X‐Ray Absorption near Edge Structure Analysis of Heavy Metals in Micro‐organism

       Changling Lao1,2,3, Liqiang Luo2, Yating Shen2, and Shuai Zhu2

       1 Guilin University of Technology, Guilin, China

       2 National Research Center of Geoanalysis, Beijing, China

       3 China University of Geosciences (Beijing), Beijing, China

5.1 Introduction

      With the ever‐increasing exploitation of mineral resources, heavy metal pollution has led to a serious effect on the ecosystem. Micro‐organisms which participate in nearly all biochemical reactions exist widely in the environment. They play an important role in the dissolution, migration, transformation, and precipitation of heavy metals. High concentrations of heavy metals possess toxic effects on micro‐organisms, including the disruption of cell membranes, alteration of enzyme specificity and protein structure, and the sometimes fatal destruction of DNA [1]. In environments, heavy metals contaminate some micro‐organisms, which are detoxified through biological processes such as adsorption, accumulation, and biotransformation, and further influence the circulation of elements and chemical species. X‐ray fluorescence (XRF) and X‐ray absorption spectroscopy (XAS) develop alongside synchrotron (SR) technology, which can be applied to understand the distribution, migration, and transformation of heavy metals.

5.2 Effects of Heavy Metals on Microbial Growth

      The biogeochemical cycle of elements will affect the growth of micro‐organisms. Some metals are essential nutrients for the growth of micro‐organisms (such as Mg, Na, Fe, Co, Cu, Mo, Ni, W, V, and Zn) [2]. Some metal elements can get energy for the metabolism of micro‐organisms through the redox reaction process (such as As(III/V), Fe(II/III), Mn(II/IV), V(IV/V), Se(IV/VI), U(IV/VI)) [3]. There are also some heavy metal ions (such as Ag⁺, Hg²⁺, Cd²⁺, Co²⁺, CrO₄ ²⁺, Cu²⁺, Ni²⁺, Pb²⁺, and Zn²⁺) which possess a great affinity to thiol‐containing groups, and thus can easily replace essential metal ions that are typically present and necessary in many enzymes [4, 5].

The composition of the microflora is governed by their physical