Author: Amit Patel and
Jonathan Sweedler
(University of Illinois at Urbana-Champaign)


Author: Elena Rosini
(Università degli Studi dell'Insubria)
The D-AAs are a class of signaling molecules which are largely understudied. The discovery and characterization of D-AAs in living systems necessitates the development, improvement and application of an assortment of analytical methods. Since the initial measurement of D-AAs in animals, advances in approaches for D-AA analysis have been numerous – including the development of capillary electrophoresis approaches with sufficient sensitivity to target sub-cellular samples, liquid chromatography approaches which simultaneously target many D-AAs and imaging approaches to uncover the localization of these molecules. These measurement approaches, when paired with appropriate experiment design and established animal models, have led to a variety of discoveries (including the characterization of novel enzymes, the establishment of two D-AAs as “classical” transmitters and a better understanding of many D-AAs in the nervous and endocrine systems of many animals).
Capillary Electrophoresis (CE)

Capillary Electrophoresis (CE) is a separation technique which leverages the differences in ionic mobility of solvated ions in the presence of an applied electric field, where ionic mobility is dependent on the charge and stokes radius of the ions. CE has proven useful for the analysis of a variety biomolecules, ranging from single monosaccharides up to entire proteins. Relative to other separation techniques, such as liquid chromatography and gas chromatography, CE has a number of notable advantages — high efficiency, short separation times, and low sample volume requirements.  The requirement for low sample volumes has allowed CE to characterize the DAAs in individual neurons.
In order to discriminate between the L- and D-forms of amino acids, chiral selectors are commonly employed. The differential interaction between the chiral forms of the analytes and the chiral selector result in their separation. Alternatively, CE without the use of such chiral selectors can also be used to separate and measure the amount of each enantiomer provided they are first reacted with an optically pure chiral reagent (resulting in the formation of diastereomers prior to analysis).
Capillary electrophoresis can be paired with a variety of detection modalities for D-AA analysis, where laser-induced fluorescence (LIF) and mass spectrometry (MS) are two of the more common means of detection. The employment of the aforementioned detection modalities provides sufficient sensitivity to enable the analysis of D-AAs in spite of limited sample volumes; yes, these analyses can even be carried out at the subcellular level!

Electropherograms resulting from the subcellular analysis of the processes and cell soma of a single neuron from Aplysia.
Adapted from Miao, H., et al. J. Neurochem., 2006, 97(2), 595-606.
Liquid Chromatography (LC)

Since its advent, liquid chromatography (LC) has been a separation technique of choice for many analytical chemists. LC is well known as a robust approach which separates different chemical species on the basis of different retention times on a separation column as a consequence of differential interactions between the analytes, the liquid mobile phase and the stationary phase. Much like the case described with CE, LC can discriminate on the basis of chirality by the employment of a chiral stationary phase, the incorporation of additives which affect the retention time or by reacting the chiral species with an optically pure chiral reagent. Many of the early applications of LC for the analysis of D-AAs employed fluorescence as its detection modality. This was done in part to take advantage of the sensitivity afforded by fluorescence detection, which was useful since these analyses were targeting what are often low abundance analytes. The use of fluorescence detection was also employed because the precolumn derivatization also generated diasteroisomers. There are many examples of mass spectrometry being used as the detection modality for LC because of its sensitivity and selectivity.

In situ hybridization (ISH)

ISH is an approach which enables the localization of a given sequence of nucleic acids (RNA/DNA) in tissue. This is generally accomplished through the application of a probe strand of nucleic acids which is complementary to the target strand, and labeled in some way to enable observations of the target localization in the tissue of interest. In this way, RNA or DNA corresponding to proteins connected to D-AAs, such as those related to known racemases or D-form amino acid oxidases, can be visualized.

ISH demonstrating the distribution of serine racemase expression in an adult mouse brain slice. Image acquired from the Allen Brain Atlas. (accessed Dec 29, 2016).
Immunohistochemistry (IHC)

When interested in studying the localization of proteins associated with the D-AAs (such as a racemase responsible for the synthesis of a given D-AA), fluorescence imaging following sample treatment with antibodies is often employed. Where the ISH approach above targets strands of nucleic acids, this approach is generally used to measure proteins that have been translated - avoiding staining of cells/regions which contain the corresponding DNA/RNA but not the translated proteins. Shown below is a whole mount immunohistochemical stain of the D-aspartate racemase (DAR1) in Aplysia californica.

DAR1 immunoreactivity localization in the cerebral ganglion of Aplysia californica.
Image adapted from Wang, L., et al. J. Biol. Chem., 2011, 286(15), 13765–13774.
Localization of Enzyme Activity

Where ISH can localize specific strands of nucleic acids and IHC can be used to image translated proteins one can also treat tissue with carefully selected reagents to image on the basis of enzyme activity. This was eloquently demonstrated by Sasabe and coworkers with D-amino acid oxidase (DAO) in mouse tissue (shown below).

Visualization of DAO activity in the human cerebellum.
Note the higher levels of activity in molecular layer (Mol) relative to the granular layer (Gr) and minimal activity in the white matter (WM).
Image adapted from Sasabe, J. et al. Front Synaptic Neurosci., 2014, 6(14).
What are Biosensors?
The term “biosensor” is short for “biological sensor”, a chemical sensing analytical device which converts a biological response into an electrical signal.
The device is made up of a transducer and a biological element (bioreceptor) that may be an enzyme, an antibody or a nucleic acid. The bioreceptor interacts with the analyte being tested and the biological response is converted by the transducer into an electrical, optical or thermal signal. The final result is a display depicting the presence of the target analyte (Turner et al., 1987).

The key element of a biosensor is the transducer which makes use of a physical change accompanying the reaction. This may be:
  • the heat output by the reaction (calorimetric biosensors)
  • changes in the distribution of charges producing an electrical potential (potentiometric biosensors)
  • movement of electrons produced in a redox reaction (amperometric biosensors)
  • light output or a light absorbance/fluorescence change during the reaction (optical biosensors)
  • effect due to the mass of the reactants or products (piezo-electric biosensors)
A successful biosensor must possess the following beneficial features:
  • the biocatalyst must be highly specific for the purpose of the analyses, be stable under operational conditions and show good stability over a large number of assays (i.e. >> 100);
  • the reaction should be independent of physical parameters as stirring, pH and temperature;
  • the response should be accurate, precise, reproducible, linear and free from (electrical) noise;
  • the complete biosensor should be cheap, small, portable and capable of being used by semi-skilled operators.
Biosensor applications
The commercial application of biosensors had a significant impact in a number of areas. The market is comprised of different segments (namely: medical, environmental, and food), with medical applications being to dominant player (Hall, 1986). Biosensors represent a rapidly expanding field, with an estimated 60% annual growth rate; the major impetus coming from the health-care industry. The estimated world analytical market is about $ 12,000,000,000/year (30% is in the health care area): there is clearly a vast market expansion potential as less than 0.1% of this market is currently using biosensors. The demand for reliable, inexpensive and rapid methods for assessment of quality will increase.
Detection of selected D-amino acids (and derivatives)
The presence of D-amino acids in biological samples can be detected using different analytical methods, such as capillary gas chromatography, reversed-phase HPLC and capillary electrophoresis, which often are time-consuming, expensive and not suitable for on-line application. A rapid and selective determination of D-amino acids (and derivatives) may have an important impact on life sciences at different levels. In details:
  • Safety and quality of food: the total content of D-amino acids is considered a parameter for assessing the quality of food as high levels indicate microbial growth (Rosini et al., 2008);
  • Environmental sector: the detection in water samples of glyphosate, a broad spectrum herbicide widely used in the world, is of utmost importance (Pedotti et al., 2009);
  • Human health: detection of D-aspartate (involved in the pathophysiology of infertility and in the regulation of brain functions) and D-serine (associated with various diseases, such as Alzheimer's and amyotrophic lateral sclerosis and disorders of the nervous system such as schizophrenia and bipolar disorder). Among amino acids and amine, glycine (involved in the pathophysiology of schizophrenia and other neurological diseases, see Rosini et al., 2014a), histamine (indicated in the diagnosis of diseases related to its release, the identification of clinical situations and in defining the mechanisms of adverse reactions to drugs, see Rosini et al., 2014b), and sarcosine (proposed as a novel biomarker for prostate cancer, see Rosini et al., 2014a).
Amperometric biosensors have been developed, based on the detection of hydrogen peroxide or ammonia produced by the D-amino acid oxidase (DAAO) reaction. A chiral analysis of different amino acids was performed by using a composite electrode. DAAO, horseradish peroxidase (HRP) and the mediator ferrocene were co-immobilized by simple physical inclusion into the bulk of a graphite-Teflon electrode matrix (Dominguez et al., 2001). Various immobilization procedures were investigated for the co-immobilization of HRP and DAAO in carbon paste electrodes, using just plain adsorption, adsorption with addition of PEI, glutaraldehyde, or the combination of glutaraldehyde and PEI. A stable response was obtained only by the combination of adsorption and PEI (0.1%), and a ratio DAAO:HRP 2:1 (Kacaniklic et al., 1994). A rapid measurement of D-amino acids was achieved by screen-printed amperometric biosensors incorporating immobilized DAAO and rhodinised carbon to facilitate hydrogen peroxide oxidation (Sarkar et al., 1999). The device responded to all common D-amino acids, the exception being D-proline, and exhibited stability over a 56 days period. A home-made bioreactor was built into a FIA system with DAAO immobilized in a thin-layer Plexi-cell; the working concentration range was between 0.2-3 mM, showing a standard deviation of 2.7% (Varadi et al., 1999). All these sensors showed a broad substrate specificity since they were based on the activity of DAAO from pig kidney, and they do not solve the need for a simple and cheap measuring device. A quickly and selective detection of D-amino acids content in biological samples was achieved by mean of a simple amperometric biosensor using wild-type RgDAAO adsorbed on the graphite electrode (Sacchi et al., 1998). The sensor is characterised by a proportional response between 0.2-3 mM D-alanine and shows a linear response starting from 100 µM D-alanine. Noteworthy, a current response was obtained for the D-amino acids with an acidic side chain, when the M213R RgDAAO variant was added in the reaction chamber (Sacchi et al., 2004). The total D-amino acids content in food specimen was assayed by an inexpensive, simple and rapid device in which the D-amino acid composition does not alter the results. A screen-printed electrode amperometric biosensor based on a mixture of RgDAAO variants was used; the response was independent from the composition of the solution, with a detection limit of 0.25 mM and a mean response time of 10-15 min (Rosini et al., 2008). Notably, a microbiosensor based on cylindrical platinum microelectrode covered with a membrane of poly-m-phenylenediamine and a layer of immobilized DAAO from Rhodotorula gracilis (RgDAAO) was developed to monitor D-serine levels in vivo (Pernot et al., 2008); a detection limit of 16 nM and a mean response time of 2 s were achieved.

Fluorimetric assays

In this context, we set up two biosensors based on different, selected enzyme variants of the flavoenzyme glycine oxidase. The system is based on a simple fluorimetric assay, characterized by low cost and ease of use; the fluorescence intensity emission of a commercial dye transducer is proportional to the concentration of analyte (Rosini et al., 2014a). In real time the optical sensing system assays glycine or sarcosine in biological samples with a detection limit ≤ 0.5 µM. The glycine concentration detected in U87 human glioblastoma cell extracts is in good agreement with the value obtained by using the reference HPLC method (7.5 versus 6.7 µM, respectively); interestingly, the assay allowed to quantify the glycine concentration in human plasma, in good agreement with the values reported in the literature.
References for "Measurement of D-AAs: Abundance":

Capillary Electrophoresis (CE)
  • Miao, H., Rubakhin, S.S., Scanlan, C.R., Wang, L., Sweedler, J.V., D-Aspartate as a putative cell-cell signaling molecule in the Aplysia californica central nervous system, J. Neurochem., 2006, 97(2), 595-606.
  • Ota, N., Rubakhin, S.S., Sweedler, J.V., D-Alanine in the islets of Langerhans of rat pancreas, BBRC, 2014, 447, 328–333.
  • Otsuka, K., Karuhaka, K., Higashimori, M., Terabe, S. Optical resolution of amino acid derivatives by micellar electrokinetic chromatography with N-dodecanoyl-L-serine. J. Chromatogr., 1994, 680, 317–20.
  • Scanlan, C., Shi, T., Hatcher, N., Rubakhin, S.S., Sweedler, J.V., Synthesis, accumulation, and release of D-aspartate in the Aplysia californica central nervous system, J Neurochem., 2010, 115(5), 1234–1244.
Liquid Chromatography (LC)
  • Dunlop, D.S., Neidle, A. The separation of D/L amino acid pairs by high-performance liquid chromatography after precolumn derivatization with optically active naphthylethyl isocyanate. Analytical Biochemistry, 1987, 165(1), 38-44.
  • Dunlop, D.S., Neidle, A., McHale, D., Dunlop D.M., Lajtha, A. The presence of free D-aspartic acid in rodents and man, BBRC, 1986, 141(1), 27-32.
  • Kimura, T., Hamase, K., Miyoshi, Y., Yamamoto, R., Yasuda, K., Mita, M., Rakugi, H., Hayashi, T., Isaka, Y. Chiral amino acid metabolomics for novel biomarker screening in the prognosis of chronic kidney disease. Scientific Reports, 2016, 6, Article number: 26137.
Liquid Chromatography (LC)
  • Dunlop, D.S., Neidle, A. The separation of D/L amino acid pairs by high-performance liquid chromatography after precolumn derivatization with optically active naphthylethyl isocyanate. Analytical Biochemistry, 1987, 165(1), 38-44.
  • Dunlop, D.S., Neidle, A., McHale, D., Dunlop D.M., Lajtha, A. The presence of free D-aspartic acid in rodents and man, BBRC, 1986, 141(1), 27-32.
  • Kimura, T., Hamase, K., Miyoshi, Y., Yamamoto, R., Yasuda, K., Mita, M., Rakugi, H., Hayashi, T., Isaka, Y. Chiral amino acid metabolomics for novel biomarker screening in the prognosis of chronic kidney disease. Scientific Reports, 2016, 6, Article number: 26137.
References for "Measurement of D-AAs: Localization":

Immunohistochemistry (IHC)
  • Wang, L., Ota, N., Romanova, E.V., Sweedler, J.V. A novel pyridoxal 5’-phosphate-dependent amino acid racemase in the Aplysia californica central nervous system. J. Biol. Chem., 2011, 286(15), 13765–13774.
Localization of Enzyme Activity
  • Sasabe, J., Suzuki, M., Imanishi, N., Aiso, S. Activity of D-amino acid oxidase is widespread in the human central nervous system. Front Synaptic Neurosci., 2014, 6(14).
References for "Sensing and Dynamic Measurements":

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  • Hall E.A.H. “The developing biosensor arena” (1986) Enzyme Microb Technol 8: 651-658
  • Kacaniklic V., Johansson K., Marko-Varga G., Gorton L., Jönsson-Pettersson G., Csöregi E. “Amperometric biosensors for detection of L- and D-amino acids based on coimmobilized peroxidase and L- and D-amino acid oxidases in carbon paste electrodes” (1994) Electroanalysis 6: 381-390
  • Pedotti M., Rosini E., Molla G., Moschetti T., Savino C., Vallone B., Pollegioni L. “Glyphosate resistance by engineering the flavoenzyme glycine oxidase” (2009) J Biol Chem 284: 36415-36423
  • Pernot P., Mothet J.P., Schuvailo O., Soldatkin A., Pollegioni L., Pilone M., Adeline M.T., Cespuglio R., Marinesco S. “Characterization of a yeast D-amino acid oxidase microbiosensor for D-serine detection in the central nervous system” (2008) Anal Chem 80: 1589-1597 
  • Rosini E., Molla G., Rossetti C., Pilone M.S., Pollegioni L., Sacchi S. “A biosensor for all D-amino acids using evolved D-amino acid oxidase” (2008) J Biotechnol 135: 377-384
  • Rosini E., Piubelli L., Molla G., Frattini L., Valentino M., Varriale A., D’Auria S., Pollegioni L. “Novel biosensors based on optimized glycine oxidase” (2014a) FEBS J 281: 3460-3472
  • Rosini E., Tonin F., Vasylieva N., Marinesco S., Pollegioni L. “Evolution of histamine oxidase activity for biotechnological applications” (2014b) Appl Microbiol Biotechnol 98: 739-748
  • Sacchi S., Pollegioni L., Pilone M.S., Rossetti C. “Determination of D-amino acids using a D-amino acid oxidase biosensor with spectrophotometric and potentiometric detection” (1998) Biotechnol Tech 12: 149-153
  • Sacchi S., Lorenzi S., Molla G., Pilone M.S., Rossetti C., Pollegioni L. “Engineering the substrate specificity of D-amino-acid oxidase” (2004) J Biol Chem 277: 27510-27516
  • Sarkar P., Tothill I.E., Setford S.J., Turner A.P. “Screen-printed amperometric biosensors for the rapid measurement of L- and D-amino acids” (1999) Analyst 124: 865-870
  • Turner A.P.F., Karube I., Wilson G.S. “Biosensors: fundamentals and applications” (1987) Oxford, U.K.: Oxford University Press Váradi M., Adányi N., Szabó E.E., Trummer N. “Determination of the ratio of D- and L-amino acids in brewing by an immobilised amino acid oxidase enzyme reactor coupled to amperometric detection” (1999) Biosens Bioelectron 14: 335-340