Real-time fluorescence detection of Hg²⁺ ions with high sensitivity by exponentially isothermal oligonucleotide amplification

Abstract

A new sensitive assay for Hg²⁺ ions has been demonstrated in this work based on isothermal exponential amplification reaction (EXPAR). In the presence of Hg²⁺ ions, a thymine (T)-rich oligonucleotide probe will firstly combine with a T-rich template through Hg²⁺-mediated T–Hg²⁺–T interaction and subsequently trigger the EXPAR reaction. EXPAR is a simple, fast and highly efficient amplification technique, which can provides 10⁶–10⁹ fold amplification under isothermal conditions within minutes. Therefore, with real-time fluorescent detection of EXPAR products triggered by Hg²⁺ ions, high sensitivity is achieved and as low as 100 pM Hg²⁺ ions can be accurately determined in a 10 μL reaction system. Furthermore, due to the specific binding between Hg²⁺ and thymine, even 10–100 fold excess of other metal ions do not interfere with the detection of Hg²⁺ ions, showing high specificity of this proposed assay.

---

Introduction

As is well known, mercury pollution is a global problem. Through mercury-contaminated natural waters and foods, mercury can be accumulated in the human body and lead to damage to the brain, nervous system, etc. Hg²⁺ ion detection is very important for monitoring mercury pollution and preventing mercury poisoning. Recently, intense research has been carried out worldwide into the development of Hg²⁺ ion detection methods.¹ Many new small-molecules have been synthesized and characterized as fluorescent² and colorimetric³ probes for Hg²⁺ ion detection over the past several years. However, these methods for Hg²⁺ ion detection suffer from the disadvantages of low water solubility of the small-molecule probe, cross-response by other heavy metal ions, and relatively low sensitivity, in which the detection limits are generally in the low μM range. Other strategies for Hg²⁺ ion detection based on proteins,⁴ CdTe and InP nanocrystals,⁵ carbon nanotubes,⁶ Ag nanocluster,⁷ gold nanorods and nanoparticles⁸ have been also reported.

It has been well documented that Hg²⁺ ions can strongly bind to thymine–thymine (T–T) base pairs in DNA duplexes.⁹ Remarkable progress in the design of Hg²⁺ ion-responsive sensors based on T–Hg–T interaction has been recently achieved because these sensors are highly selective for Hg²⁺ ion detection and can be operated in aqueous solution at neutral pH. Firstly, a mercury specific DNA (MSD) has been designed for Hg²⁺ ion detection. MSD is a T-rich oligonucleotide which presents a random coil form in absence of Hg²⁺ ions and forms a hairpin structure in the presence of Hg²⁺ ions.¹⁰ The Hg²⁺ ion-induced conformational changes of MSD have been detected by fluorescence resonance energy transfer (FRET),⁹ quantum dot,¹¹ conjugated polymers,¹² and gold nanoparticle aggregation.¹³ The second strategy for Hg²⁺ ion detection has been developed based on the hybridization between DNA probes containing T–T mismatched base pairs by using DNA-functionalized gold nanoparticles as the signalling units. These detection methods include melting temperature-based colorimetry,¹⁴ and chip-based scanometric detection.¹⁵ The methods based on T–Hg–T interaction have improved the sensitivity for Hg²⁺ ion determination. The detection limits of these methods are greater than or equal to 10 nM. Interest is rapidly emerging in the exploration of highly sensitive determination of Hg²⁺ ions, such as electrochemical detection,¹⁶ DNAzyme-based method,¹⁷ capillary electrophoresis,¹⁸ fluorescence method,^10,19 which push the detection limits for Hg²⁺ ion down to a few nanomolar.

I. Willner group has demonstrated a new strategy for highly sensitive determination of Hg²⁺ ions by using a DNA-based machine as molecular system to amplify the Hg²⁺-responsive readout signal based on the combination of polymerase strand extension and specific cleavage with nicking enzyme.¹³ However, the signal amplification is a linear amplification. Thus, the sensitivity for Hg²⁺ ion determination is not very high (with detection limit of 1.0 nM) and the amplification needs a long time (1 h). In this paper, we report a highly sensitive method for Hg²⁺ ion determination based on exponential amplification reaction (EXPAR),²⁰ which can provides 10⁶–10⁹ fold amplification under isothermal conditions within minutes. With real-time fluorescent detection of EXPAR products triggered by Hg²⁺ ions, as low as 100 pM Hg²⁺ ions can be accurately determined and the dynamic range is over 2 orders of magnitude. Moreover, this method does not require any DNA label and real-time detection saves all post-reaction steps. The fast and isothermal EXPAR results in simple and rapid detection procedure.

Results and discussion

The principle of the EXPAR-based Hg²⁺ ion assay

The principle of the EXPAR-based Hg²⁺ ion assay is illustrated in Fig. 1a. We first prepare the specific DNA probe X and the amplification templates of X′–Y′ and Y′–Y′ (see the sequences in Fig. 1b). The template X′–Y′ contains the sequence X′ (5′-gctttttttttt-3′) and Y′ (5′-cctacgactgg-3′), which are separated by nine bases (5′-aacaGACTC-3′). The T bases in probe X can combine with T bases in X′ based on the strong T–Hg²⁺–T interactions only when Hg²⁺ ions are present. The bases of GC in the 3′-terminus of probe X, therefore, can hybridize with CG bases in the template X′–Y′. Subsequently, probe X can extend along the template X′–Y′ in the presence of DNA polymerase and dNTPs and produce DNA strand of 5′-GAGTCtgtt-3′ and 5′-ccagtcgtagg-3′ (defined as Y, which is complementary to Y′). The sequence of 5′-GAGTC-3′ is the recognition site of nicking enzyme which can cleave the DNA strand at the fourth base downstream from the recognition site. After the cleavage, the DNA strand containing the recognition site will extend again and the short DNA strand Y will be displaced and released. After that, the extension, cleavage and strand displacement can be continuously repeated to release more DNA strand Y, which can hybridize with the amplification template Y′–Y′ at its 3′-terminus and extend along the template. The template Y′–Y′ contains two repetitive Y′ sequences at 3′- and 5′-terminus, respectively, where the two repeat Y′ sequences are also separated by nine bases which are the same as mentioned above. According to same principle demonstrated above, the extension of the DNA strand Y along the template Y′–Y′ can continuously produce more and more DNA strand Y by combination of polymerase strand extension and single strand nicking. The products of the DNA strand Y will hybridize with other template Y′–Y′ and then produce more and more DNA strand Y, and so on, giving rise to chain reaction and resulting in EXPAR. With the EXPAR-based amplification, a small number of Hg²⁺ ions can produce a large amount of double strand (ds) DNA. SYBR Green I (SG), which is by far the most sensitive reagent for staining dsDNA,^10,21 is utilized as the fluorescent dye for real-time detection of the EXPAR products.

Fig. 1 (a) Schematic representation of the EXPAR with Hg²⁺ ions as the trigger. (b) Sequences of the amplification templates. P indicates a phosphate group.

Optimization of experimental conditions for Hg²⁺ ion detection

In the EXPAR-based homogeneous assay, the reaction conditions, such as concentrations of Vent (exo⁻) DNA polymerase and Nt. BstNBI nicking enzyme, have important effect on Hg²⁺ ion detection. To investigate the influence of the amount of DNA polymerase used in EXPAR reaction on Hg²⁺ ion detection, the real-time fluorescence curve produced by 50 nM Hg²⁺ ions was measured by using 0.04 U μL⁻¹, 0.06 U μL⁻¹, 0.08 U μL⁻¹, and 0.10 U μL⁻¹ Vent (exo⁻) DNA polymerase. The blank were treated in the same way for EXPAR without Hg²⁺ ions. As shown in Fig. 2, when the amount of DNA polymerase increases, the time at which the fluorescence signals for both Hg²⁺ ions and blank are detectable is gradually shortened. Fig. 2b shows the maximum interval of the point of inflection (POI, the time corresponding to the maximum slope in the fluorescence curve) between Hg²⁺ and the blank. Therefore, 0.06 U μL⁻¹ Vent (exo⁻) DNA polymerase is considered to be optimum amount used in the EXPAR reaction.

Fig. 2 The influence of the amount of Vent (exo⁻) DNA polymerase on the real-time fluorescence curve produced by 50 nM Hg²⁺ ions with EXPAR reaction. The final concentration of Vent (exo⁻) DNA polymerase is (a) 0.04 U μL⁻¹, (b) 0.06 U μL⁻¹, (c) 0.08 U μL⁻¹, and (d) 0.1 U μL⁻¹. The others' final concentrations: [probe X] = 0.1 μM, [template X′–Y′] = 0.1 μM, [template Y′–Y′] = 0.4 μM, [each dNTP] = 250 μM, [Nt. BstNBI] = 0.8 U μL⁻¹, [SYBR Green] = 0.4 μg mL⁻¹. The EXPAR reaction and measurement of fluorescence are carried out according to the procedure described in the Experimental section.

Fig. 3 shows the influence of the amount of nicking endonuclease used in EXPAR reaction on the real-time fluorescence curve produced by 50 nM Hg²⁺ ions and the blank. As shown in Fig. 3, when the amount of nicking enzyme increases, the time at which the fluorescence signals are detectable for both Hg²⁺ ions and blank is gradually increased. Fig. 3c shows the maximum interval of POI between Hg²⁺ ions and the blank. Therefore, 0.8 U μL⁻¹ Nt. BstNBI nicking endonuclease was selected for the Hg²⁺ ion detection.

Fig. 3 The influence of the amount of nicking endonuclease used in the EXPAR reaction on the real-time fluorescence curve produced by 50 nM Hg²⁺ ions. The amount of Nt. BstNBI nicking endonuclease is (a) 0.4 U μL⁻¹, (b) 0.6 U μL⁻¹, (c) 0.8 U μL⁻¹, and (d) 1.0 U μL⁻¹. Other experiment conditions are the same as described in Fig. 2 except the amount of Vent (exo⁻) DNA polymerase is 0.06 U μL⁻¹.

Hg²⁺ ion detection

Under the optimum conditions, as a proof-of-principle experiment, the real-time fluorescence signals produced by Hg²⁺ ions with different concentrations were measured by using 100 nM probe X. As shown in Fig. 4, POI values are well linearly depended on logarithm (lg) of Hg²⁺ ion concentration in the range from 1.0 nM to 50.0 nM. The correlation equation is POI = 25.62–0.75 lg C_Hg (R = −0.9934).

Fig. 4 (a) Real-time fluorescence curves for the EXPAR triggered by Hg²⁺ ions. (b) Relationship between the POI value and the logarithm of the amount of Hg²⁺ ions. Final concentrations: [probe X] = 100 nM, [template X′–Y′] = 0.1 μM, [template Y′–Y′] = 0.4 μM, [each dNTP] = 250 μM, [Vent (exo⁻) DNA polymerase] = 0.06 U μL⁻¹, [Nt. BstNBI] = 0.8 U μL⁻¹, [SYBR Green] = 0.4 μg mL⁻¹. The error bars were estimated from the standard deviation of three repetitive measurements.

It is worth noting that the sensitivity for Hg²⁺ ion detection can be greatly improved by reducing the amount of probe X used for the EXPAR. As demonstrated in Fig. 5, when 10 nM probe X was used, as low as 100 pM Hg²⁺ ions can be well detected. There is a good linear relationship between POI valves and lg of Hg²⁺ ion concentrations in the range of 100 pM–10 nM. The correlation equation is POI = 20.91–1.17 lg C_Hg (M) and the correlation coefficient R is −0.9950.

Fig. 5 (a) Real-time fluorescence curves for the EXPAR triggered by Hg²⁺ ions. (b) Relationship between the POI value and the logarithm of the amount of Hg²⁺ ions. Final concentrations: [probe X] = 10 nM, the other experiment conditions are the same as those for Fig. 4. The error bars were estimated from the standard deviation of three repetitive measurements.

The probe X contains 10 T bases. We suppose that the formation of stable complex between probe X and the template X′–Y′ needs more than one Hg²⁺ ion. Therefore, when probe X concentration is high (such as 100 nM), the probability of that one probe X molecule combines several Hg²⁺ ions become too small to form stable complex between probe X and template X′–Y′ when Hg²⁺ ions are at low concentration (such as less than 1.0 nM). Therefore, Hg²⁺ ions at the concentration less than 1.0 nM can not initiate the EXPAR. Reducing the probe X to 10 nM can increase the probability of that one probe X molecule combines several Hg²⁺ ions at low concentration of Hg²⁺ ions. Therefore, the sensitivity for Hg²⁺ ion detection can be improved. However, as Fig. 6 shown, reducing the concentration of probe X down to 1 nM, the sensitivity for Hg²⁺ ion detection can not be further improved, where the lowest concentration of Hg²⁺ ions is still 100 pM.

Fig. 6 The real-time fluorescence curves for the EXPAR triggered by Hg²⁺ ions. The other experiment conditions are the same as those for Fig. 4 except the concentration of DNA probe X, which is 1 nM.

As specific DNA probes containing T-rich and C or G bases are usually used for Hg²⁺ ion detection,^10,13,19 we have also designed the DNA probe X₂ (5′-ccttcttcttgc-3′) and corresponding amplification template X′₂–Y′ (5′-cctacgactggaacagactcgcttgttgttgg-3′) for Hg²⁺ ions detection based on EXPAR.

Fig. 7 shows the results of Hg²⁺ ions detection when the sequences of DNA probe and corresponding amplification template were changed. We obtained the same results as that using probe X and amplification template X′–Y′. At the concentration of 100 nM DNA probe X₂, the lowest concentration of detected Hg²⁺ ions is 1.0 nM (Fig. 7a). Reducing the concentration of the DNA probe X₂ down to 10 nM, 100 pM Hg²⁺ ions could be detected (Fig. 7b).

Fig. 7 The real-time fluorescence curves of Hg²⁺ ions using DNA probe X₂. The EXPAR reaction and measurement of fluorescence were carried out according to the procedure described in the Experimental section except the concentration of DNA probe X₂, which is used as (a) 100 nM, (b) 10 nM, respectively.

Selectivity of the Hg²⁺ assay

To evaluate the specificity of the proposed Hg²⁺ ion assay, the interference of other metal ions, including Ca²⁺, Al³⁺, Mn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Ag⁺, Cd²⁺, and Pb²⁺ were investigated. Under the optimum conditions, the real-time fluorescence signal produced by Hg²⁺ ions could be separated completely from those produced by the other metal ions. From Fig. 4b, the correlation equation for Hg²⁺ ion determination is POI = 25.62–0.75 lg C_Hg. We suppose the POI values produced by Hg²⁺ ions and other metal ions are POI_Hg and POI_M, respectively. C_Hg and C_M are the concentrations corresponding to POI_Hg and POI_M. Therefore, C_M/C_Hg (%) is defined as the relative detection, which can be used to estimate the interference of other metal ions for Hg²⁺ ion determination. According to the correlation equation:

POI_M − POI_Hg = −0.75(lg C_M − lg C_Hg) (1)

lg(C_M/C_Hg) = −(POI_M − POI_Hg)/0.75 (2)

According to the eqn (2), the interference of various metal ions for Hg²⁺ ion determination is respectively calculated and the results are shown in Fig. 8. One can see from Fig. 8 that the interference of Cu²⁺, Pb²⁺, Al³⁺, Cd²⁺, Ag⁺ ions is estimated to be 7.8%, 4.7%, 2.6%, 1.8% and 1.7%, respectively. All interference produced by other metal ions is less than 1.0%.

Fig. 8 Selectivity of the Hg²⁺ ion assay over other metal ions. The concentration of Hg²⁺ ions is 50 nM, and the other metal ions are 10.0 μM except Cu²⁺, Zn²⁺, Ag⁺, Cd²⁺ and Pb²⁺, which concentrations are respectively at 1.0 μM. The concentration of DNA probe X is 100 nM. Other conditions were the same as those described in Experimental section.

Detection of the potable water sample

The EXPAR-based method has been used to test potable water samples. As shown in Fig. 9, the amount of Hg²⁺ ions in the potable water are too small to be detectable. By addition small amounts (0.5 nM and 1.0 nM) of Hg²⁺ ions in the potable water, the well defined signals can be detected. The average amounts of Hg²⁺ ions determined from five repetitive measurements are 0.56 nM and 1.07 nM, respectively. The analytical recoveries are 112% and 107%, respectively. Therefore, the proposed method may be used to determine Hg²⁺ ions in the water samples.

Fig. 9 Detection of Hg²⁺ in potable water sample. 1.0 μL potable water was added to the EXPAR reaction mixture with the final volume of 10 μL. [probe X] = 10 nM. The EXPAR and real-time measurement of fluorescence intensity was carried out according to the experimental procedure described in Experimental section.

Conclusions

In summary, we have developed a new assay of Hg²⁺ ions with high sensitivity and selectivity. This method bases on the strong T–Hg–T interaction and uses EXPAR reaction as the amplification technique to detect Hg²⁺ ions in aqueous solution. This detection strategy is simple, fast and cost-effective. It is without any labels and can detect 100 pM Hg²⁺ ions. Its high selectivity allows detection of Hg²⁺ ions in the presence of an excess (20–200-fold) of other metal ions. The method has potential for detection of Hg²⁺ ion in water pollution, food safety. In this work, only detection of free Hg²⁺ ions is studied. The detection of speciation of mercury is very important for monitoring mercury pollution and bioavailability. Therefore, the interactions between thymine–thymine bases and different speciation of mercury and the detection of speciation of mercury need further study.

Experimental section

Materials and apparatus

Mercury(II) perchlorate trihydrate (Hg(ClO₄)₂·3H₂O) is purchased from Alfa Aesar China (Tianjin) Co., Ltd. Perchloric acid (HClO₄) is obtained from Dong-Fang chemical plant (Tianjin, China). PAGE-purified DNA is obtained from TaKaRa Biotechnology Co. Ltd. (Dalian, China). Vent (exo⁻) DNA polymerase and Nt. BstNBI nicking endonuclease are purchased from New England Biolabs. SYBR Green I (20× stock solution in DMSO) is purchased from Xiamen Bio-Vision Biotechnology (Xiamen, China). All solutions for EXPAR reactions are prepared in deionized sterile water. The EXPAR reactions and the real-time fluorescence measurements are performed with a Step One Real-Time PCR System (Applied Biosystems, USA).

The concentration of stock Hg²⁺ ions is 10.0 mM, which is dissolved in 1% HClO₄. In order to avoid forming insoluble product with Hg²⁺ ions, we prepare the EXPAR reaction buffer by ourselves according to the component of the ThermoPol buffer and Nt. BstNBI buffer using CH₃COO⁻ to replace Cl⁻ and SO₄²⁻.

EXPAR reaction and real-time measurement of fluorescent intensity

The reaction mixtures for EXPAR reaction are separately prepared on ice as part A and part B. Part A consists of Nt. BstNBI buffer, DNA probe, amplification template X′–Y′ and Y′–Y′, dNTPs, and Hg²⁺ target. Part B consists of ThermoPol buffer, Nt. BstNBI nicking enzyme endonuclease, Vent (exo⁻) DNA polymerase, SYBR Green I, and deionized sterile water. The EXPAR reaction is carried out in 10 μL volume containing DNA probe, 0.1 μM amplification template X′–Y′, 0.4 μM amplification template Y′–Y′, 100 μM dNTPs, 0.8 U μL⁻¹ Nt. BstNBI nicking endonuclease, 0.06 U μL⁻¹ Vent (exo⁻) DNA polymerase, 0.4× SYBR Green I, 1× thermopol buffer (20 mM Tris–HAc, pH 8.8, 10 mM KAc, 10 mM NH₄Ac, 2 mM Mg(Ac)₂, 0.1% Triton X-100), and 0.5× Nt. BstNBI buffer (25 mM Tris–HAc, pH 7.9, 50 mM NaAc, 5 mM Mg(Ac)₂, and 0.5 mM DTT). The part A and part B is immediately mixed before putting into the Real-Time PCR System. The EXPAR reaction is performed at 55 °C and the real-time fluorescence intensity is monitored at intervals of 30 s.

Acknowledgements

The project is supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China (20121301120006), the Scientific Research Projects of Higher Education of Hebei Province (Z2011131), and the Natural Scientific Research Projects of Hebei University (2010Q08).

Notes and references

1. E. M. Nolan and S. J. Lippard, Chem. Rev., 2008, 108, 3443.
2. (a) X. J. Zhu, S. T. Fu, W. K. Wong, J. P. Guo and W. Y. Wong, Angew. Chem., Int. Ed., 2006, 45, 3150; (b) R. Métivier, I. Leray and B. Valeur, Chem. – Eur. J., 2004, 10, 4480; (c) G. Zhang, D. Zhang, S. Yin, X. Yang, Z. Shuai and D. Zhu, Chem. Commun., 2005, 2161; (d) X. Guo, X. Qian and L. Jia, J. Am. Chem. Soc., 2004, 126, 2272; (e) J. Y. Kwon, Y. J. Jang, Y. J. Lee, K. M. Kim, M. S. Seo, W. Nam and J. Yoon, J. Am. Chem. Soc., 2005, 127, 10107; (f) B. Liu and H. Tian, Chem. Commun., 2005, 3156; (g) W. Jiang and W. Wang, Chem. Commun., 2009, 3913.
3. (a) M. J. Choi, M. Y. Kim and S. K. Chang, Chem. Commun., 2001, 1664; (b) S. Tatay, P. Gaviña, E. Coronado and E. Palomares, Org. Lett., 2006, 8, 3857; (c) Y. Fu, H. Li and W. Hu, Eur. J. Org. Chem., 2007, 2459; (d) F. Sancenón, R. Martínez-Máñez and J. Soto, Chem. Commun., 2001, 2262; (e) M. G. Choi, Y. H. Kim, J. E. Namgoong and S. K. Chang, Chem. Commun., 2009, 3560.
4. (a) S. V. Wegner, A. Okesti, P. Chen and C. He, J. Am. Chem. Soc., 2007, 129, 3474; (b) M. Suresh, S. K. Mishra, S. Mishra and A. Das, Chem. Commun., 2009, 2496.
5. (a) B. Chen, Y. Yu, Z. Zhou and P. Zhong, Chem. Lett., 2004, 33, 1608; (b) C. Zhu, L. Li, F. Fang, J. Chen and Y. Wu, Chem. Lett., 2005, 34, 898.
6. X. Gao, G. Xing, Y. Yang, X. Shi, R. Liu, W. Chu, L. Jing, F. Zhao, C. Ye, H. Yuan, X. Fang, C. Wang and Y. Zhao, J. Am. Chem. Soc., 2008, 130, 9190.
7. W. Guo, J. Yuan and E. Wang, Chem. Commun., 2009, 3395.
8. (a) M. Rex, F. E. Hernandez and A. D. Campiglia, Anal. Chem., 2006, 78, 445; (b) C. C. huang and H. T. Chang, Anal. Chem., 2006, 78, 8332; (c) G. K. Darbha, A. Ray and P. C. Ray, ACS Nano, 2007, 1, 208.
9. A. Ono and H. Togashi, Angew. Chem., Int. Ed., 2004, 43, 4300.
10. J. Wang and B. Liu, Chem. Commun., 2008, 4759.
11. R. Freeman, T. Finder and I. Willner, Angew. Chem., Int. Ed., 2009, 48, 7818.
12. X. Liu, Y. Tang, L. Wang, J. Zhang, S. Song, C. Fan and S. Wang, Adv. Mater., 2007, 19, 1471.
13. D. Li, A. Wieckowska and I. Willner, Angew. Chem., Int. Ed., 2008, 47, 3927.
14. (a) X. Xue, F. Wang and X. Liu, J. Am. Chem. Soc., 2008, 130, 3244; (b) J. S. Lee, M. S. Han and C. A. Mikin, Angew. Chem., Int. Ed., 2007, 46, 4093.
15. J. S. Lee and C. A. Mikin, Anal. Chem., 2008, 80, 6805.
16. (a) S. J. Liu, H. G. Nie, J. H. Jiang, G. L. Shen and R. Q. Yu, Anal. Chem., 2009, 81, 5724; (b) Z. Zhu, Y. Su, J. Li, D. Li, J. Zhang, S. Song, Y. Zhao, G. Li and C. Fan, Anal. Chem., 2009, 81, 7660.
17. (a) J. Liu and Y. Lu, Angew. Chem., Int. Ed., 2007, 46, 7587; (b) J. Li, J. Yao and W. Zhong, Chem. Commun., 2009, 4962.
18. C. K. Chiang, C. C. Huang, C. W. Liu and H. T. Chang, Anal. Chem., 2008, 80, 3716.
19. B. C. Ye and B. C. Yin, Angew. Chem., Int. Ed., 2008, 47, 8386.
20. (a) J. V. Ness, L. K. V. Ness and D. J. Galas, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 4504; (b) E. Tan, B. Erwin, S. Dames, T. Ferguson, M. Buechel, B. Irvine, K. Voelkerding and A. Niemz, Biochemistry, 2008, 47, 9987.
21. S. Mondal and V. Venkataraman, J. Biochem. Biophys. Methods, 2007, 70, 773.

---