Colorimetric Strategy for Highly Sensitive and Selective Simultaneous

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A colorimetric strategy for highly sensitive and selective simultaneous detection of histidine and cysteine based on G-quadruplex-Cu(II) metalloenzyme Changtong Wu, Daoqing Fan, Chunyang Zhou, Yaqing Liu, and Erkang Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04796 • Publication Date (Web): 02 Feb 2016 Downloaded from http://pubs.acs.org on February 12, 2016

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Analytical Chemistry

A colorimetric strategy for highly sensitive and selective simultaneous detection of histidine and cysteine based on G-quadruplex-Cu(II) metalloenzyme

Changtong Wu, †,§ Daoqing Fan, † Chunyang Zhou, † Yaqing Liu,*,‡ Erkang Wang*,† †

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of

Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, China. ‡

Key Laboratory of Food Nutrition and Safety (Tianjin University of Science and

Technology), Ministry of Education, Tianjin, 300457, China. §

Department of Chemistry and Environmental Engineering, Changchun University of

Science and Technology, Changchun, Jilin, 130022, China.

*Corresponding Author E-mail: [email protected] (Y. Liu); [email protected] (E. Wang) Tel: +86-431-85262003. Fax: +86-431-85689711.

ACS Paragon Plus Environment


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ABSTRACT In this present work, we proposed a colorimetric strategy for simultaneous detection of histidine and cysteine based on G-quadruplex-Cu(II) metalloenzyme for the first time. Due to the adding of histidine or cysteine, the formation of G-quadruplex-Cu(II) metalloenzyme will be disturbed, thus the catalytic activity to TMB-H2O2 reaction is inversely proportional to the concentration of histidine or cysteine. With this strategy, the limit of detection in experimental measurement for histidine and cysteine is 10 nM and 5 nM, respectively, which are both lower than previous colorimetric arrays. With the help of NEM, cysteine is alkylated and the reaction between Cu2+ is inhibited, so the selectivity can also be guaranteed. The cost is quite low since the developed array is label free and enzyme free by using low-cost DNA and Cu2+. More importantly, the colorimetric detection operation is very simple without any further modification process.

INTRODUCTION Histidine (His) and cysteine (Cys) are two essential amino acids in natural proteins and are of great importance in many biological functions. Histidine not only works as a neurotransmitter or neuromodulator in human muscular and nervous tissue, it also serves as a regulator of metal transmission in biological systems.1,2 A low level of histidine would cause diseases like Friedreich ataxia, epilepsy, Parkinson’s disease, and the failure of normal erythropoiesis development3, while an excess of histidine may lead to symptoms of intoxication.4,5 Cysteine also plays a vital role in human


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body in protein synthesis, detoxification, metabolism, and also acts as a significant medical biomarker associated with various diseases.6-9 As for cysteine, an abnormal concentration in vivo may lead to many diseases such as slowed growth, liver damage, Alzheimer’s disease and cardiovascular disease.10,11 Thus, the quantification of histidine and cysteine is of significant importance for human healthy. Until now, a great of efforts have been devoted to develop highly sensitive and selective

method

to

detect

histidine

and

cysteine,

including

capillary

electrophoresis12-18, spectroscopy19 ，high performance liquid chromatography20-26, nuclear magnetic resonance27, mass spectrometry28,29, fluorescence analysis method11,30-38, colorimetric methods33,39-43, and electrochemical voltammetry.2,4,44 In some cases, the analytical methods suffer from a complicated operation or an expensive cost. Moreover, to the best of our knowledge, there are only several works have been reported for simultaneous detection of histidine and cysteine.31,38,45,46 DNAzyme systems can be employed in various scientific disciplines and act as stable catalytic units for the development of biosensors.47 A frequently used DNAzyme in biosensors is peroxidase-like G-quadruplex DNAzyme system that formed by hemin and G-quadruplex.48 Further, DNAzyme based analytical methods have been constructed to sensitively detect histidine or cysteine in recent years.2,4,49,50 Considering potential application, the developed strategy should be as simple as possible. Keeping that in mind, we are interested in developing a label-free colorimetric strategy for detection of histidine and cysteine. Recently, a new kind of DNA metalloenzyme was proposed with peroxidase mimetic which simply assembled



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by polymorphic GpG-Cu(II) complexes.51 What’s more, it has been reported that the G-quadruplex-Cu(II) metalloenzyme assembled with human telomeric DNA (5’-G3(TTAG3)3-3’) and Cu2+ that can be used to catalyze the enantioselective Friedel-Crafts reaction in water with excellent enantioselectivity.52 Inspired by these reports, we find G-quadruplex-Cu(II) metalloenzyme also exhibit excellent peroxidase property which can catalyze the TMB (3, 3’, 5, 5’-tetramethylbenzidine sulfate) and H2O2 reaction and produce a high absorption peak at 652 nm wavelength. Besides, as strong binders with Cu2+ 53-55, histidine and the sulfur-containing amino acid cysteine can disturb the formation of G-quadruplex-Cu(II) metalloenzyme complex, thus leading to a low catalytic activity. Inspired by the previous investigations, a label-free colorimetric strategy for simultaneous detecting histidine and cysteine with high sensitivity and selectivity is achieved on the basis of the catalytic activity of peroxidase-like G-quadruplex-Cu(II) metalloenzyme.

EXPERIMENTAL SECTION Chemicals and Reagents. The G-quadruplex sequence (5’-G3(TTAG3)3-3’) was obtained from Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). The DNA sequence was denatured in HEPES buffer (20 mM HEPES, 150 mM NaCl, pH 7.0) at 88 oC for 10 min and cooled down slowly to room temperature to form G-quadruplex

structure.

Hydrogen

peroxide

(30wt

%)

and

3,

3’,

5,

5’-tetramethylbenzidine (TMB) were purchased from J&K Scientific LTD. The amino acids were obtained from Dingguo Biotechnology Co. Ltd. (Beijing, China). All other


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chemicals were of analytical grade and used without further treatment. The water used throughout all experiments was purified through a Millipore system. Instruments. The absorption spectra were recorded on a Cary 500 Scan UV/vis Spectrophotometer (Varian, USA) using a quartz glass cuvette with 1cm path length. Circular dichroism (CD) spectra of G-quadruplex were conducted on a JASCO J-810 spectropolarimeter (Tokyo, Japan). All the experiments were carried out at room temperature. Optimize the concentration of Cu2+ ion. Different concentrations of Cu2+ were mixed with G-quadruplex for 90 min to allow the formation of G-quadruplex-Cu(II) metalloenzyme. Then the G-quadruplex-Cu(II) metalloenzyme was added into TMB-H2O2 system. An equal volume of 2 M H2SO4 was added into the solution to stop the reaction after 4 min. The absorption spectra were measured within the range of 300-600 nm. The maximum absorption peak was recorded at 452 nm wavelength. Detection of histidine and cysteine. For histidine and cysteine detection, the amino acids were premixed with Cu2+ at optimized concentration (2.5 uM) for 30 min. Then 100 nM G-quadruplex was added into the mixture and a 90 min incubation time was followed. After that, the mixture was added into TMB-H2O2 system. An equal volume of 2 M H2SO4 was added after 4 min into the solution to stop this reaction. The absorption spectra were measured after stopping.

RESULTS AND DISCUSSION Mechanism of histidine and cysteine detection. Scheme 1 outlines the



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colorimetric

strategy

for

histidine

and

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cysteine

detection

based

on

G-quadruplex-Cu(II) metalloenzyme. The G-rich DNA strand is first denatured in HEPES buffer to form an anti-parallel G-quadruplex structure (See Figure S1 in supporting information (SI)). The addition of Cu2+ leads to the formation of G-quadruplex-Cu(II) metalloenzyme complex, exhibiting high catalytic activity on the colorimetric action of TMB in the presence of H2O2 (TMB-H2O2). The amino acid, histidine or cysteine, presents strong binding affinity with Cu2+, which inhibits the catalytic activity of G-quadruplex-Cu(II) metalloenzyme. The coloric change of the sensing system is related to the concentration of the added amino acids. Evaluation on the catalytic activity of peroxidase-like G-quadruplex-Cu(II) metalloenzyme.

To

prove

the

catalytic

activity

of

peroxidase-like

G-quadruplex-Cu(II) metalloenzyme, Cu2+ is mixed with G-quadruplex to catalyze of the colorimetric reaction of TMB-H2O2 system. Learned from the absorption spectra, Figure 1(A), the absorption intensity of the TMB-H2O2 in the absence of Cu2+ (curve a) is significantly increased after adding Cu2+ (curve b), which can improve the detection sensitivity. Considering the suppress ability of histidine on the G-quadruplex-Cu(II) metalloenzyme catalyzing TMB-H2O2 system, the concentration of Cu2+ used is very critical. If the concentration of cupric ion is too low, then the detection of histidine with high concentration might not be realized, resulting in a narrow detection range. If the cupric ion is superfluous, however, more histidine would be required to binding with Cu2+, which is not in favor of reaching a low detection limit. Thus, the optimal concentration of Cu2+ is explored by adding


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different concentrations of Cu2+ into G-quadruplex with a fixed concentration. The absorption intensity increases with increasing concentration of Cu2+ and reaches a plateau once the concentration of Cu2+ is over than 2.5 uM, as shown in Figure 1(B). Here, 2.5 uM are selected for the following detection experiments. Detection of histidine and cysteine. As shown in Scheme 1, the histidine detection is based on the competitive interaction among histidine, Cu2+ and G-quadruplex. Since Cu2+ presents stronger binding affinity to histidine than to the G-quadruplex, the addition of histidine would influence the colorimetric reaction of TMB-H2O2 system. The feasibility experiment was performed following the procedures mentioned above. To improve the reaction efficiency histidine is first mixed with Cu2+ for 30 min and then G-quadruplex is added into the mixture and incubated for 90 min. Learned from Figure 2(b), the absorption intensity of TMB is greatly decreased after incubation with histidine, comparing with that in the absence of histidine, Figure 2(a). The results are ascribed to the stronger binding affinity between Cu2+ and histidine than that with G-quadruplex. The formation of G-quadruplex-Cu(II) metalloenzyme is inhibited by the added histidine, resulting in a decrease of catalytic ability for the oxidation reaction. Thus, a colorimetric strategy for label-free detection of histidine is developed according to the G-quadruplex-Cu(II) metalloenzyme catalyzing the oxidation reaction of TMB. Figure 3(A) shows the absorption spectra of the sensing system with different concentration of histidine (from 10 nM to 30 uM). With increasing concentration of histidine, the absorption signal decreases gradually. The decrease of



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absorption values (∆A452) as a function of the concentration of histidine is revealed in Figure 3(B). Here, the ∆A452 is the absorption difference of the system in the absence of target and in the presence of target with certain concentration. It is obviously that the more histidine is added, the larger absorption decrease of TMB-H2O2 appears. The decrease upon concentration becomes smaller at 15 uM. A linear relationship is recorded over the range from 10 nM to 1.0 uM (R2 = 0.998), as shown in the inset of Figure 3(B). A limit of detection (LOD) of 10 nM is obtained for the histidine detection through experimental measurements. Comparing with previous reports about colorimetric detection of histidine (see Table S1 in SI), the developed strategy presents advantages of high sensitivity, low cost and simple operation. Cysteine is a thiol-containing amino acid and can bind with Cu2+ via the strong interaction between its thiol groups.38,54 The detection mechanism is similar as that of histidine detection. Due to the stronger binding affinity between Cu2+ and cysteine than that with G-quadruplex, the more cysteine is added, the less G-quadruplex-Cu(II) metalloenzyme is generated and thus resulted in a decrease of catalytic ability on the colorimetric reaction of TMB-H2O2. Figure 3(C) depicts the relationship between absorption of TMB-H2O2 and cysteine concentration in the range of 5 nM to 20 uM. The absorption intensity gradually decreases with increasing cysteine. The absorption change values (∆A452) at 452 nm as a function of different concentration of cysteine are vividly showed in Figure 3(D). The ∆A452 signal increases with increasing cysteine and exists a good linear relationship over the concentration range from 5 nM to 500 nM (R2 = 0.998) as showed in the inset of Figure 3(D). The LOD of cysteine is


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5 nM through experimental measurements, which is more sensitive than previous reports about colorimetric detection of cysteine (see Table S2 in SI). Evaluation of the selectivity. Considering that both histidine and cysteine can be detected by the above methods, a problem is then arisen that how to distinct histidine from cysteine if both of them exists simultaneously. One possible solution is to make use of the different properties of histidine and cysteine. It is reported that NEM (N-ethylmaleimide) can cause alkylation of cysteine56 and thus can inhibit the interaction between cysteine and Cu2+. As shown in Figure 4(c), the absorption intensity of the TMB-H2O2 is greatly decreased after addition of cysteine into the G-quadruplex-Cu(II) metalloenzyme system. Once adding NEM into the sensing system, the absorption intensity of TMB-H2O2 is restored, Figure 4(b), which is close to the catalytic ability of G-quadruplex-Cu(II) metalloenzyme on TMB-H2O2, Figure 4(a). The results confirm that the influence of cysteine on the colorimetric reaction could be eliminated with the help of NEM. Thus, NEM can be used as inhibitor to help distinguishing histidine from cysteine. Distinguishing histidine and cysteine from other amino acids is further explored. Here, several kinds of amino acids with high concentration (20 uM) are used to perform the experiment. As shown in Figure 5, the absorption change at 452 nm caused by the other amino acids is much lower than that caused by histidine. The results validate that the developed strategy presents excellent selectivity and can distinguish histidine from other amino acids. The selectivity for cysteine detection is also ensured. Thus, a highly sensitive and selective colorimetric method for cysteine and histidine detection is realized in this work.



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CONCLUSIONS In summary, a colorimetric strategy for simultaneous detection of histidine and cysteine has been developed for the first time by making use of the peroxidase-like property

of

G-quadruplex-Cu(II)

metalloenzyme.

The

G-quadruplex-Cu(II)

metalloenzyme system exhibits high catalytic ability on colorimetric reaction of TMB in the presence of H2O2 which is defined as signal to quantification the concentration of histidine or cysteine. Based on the competition reaction among target (histidine or cysteine), G-quadruplex with Cu2+, a highly sensitive and selective colorimetric array for cysteine and histidine detection is achieved attributing to the use of NEM to eliminate the influence of cysteine. The detection limits for histidine and cysteine are 10 nM and 5 nM through experimental measurements, respectively. To the best of our knowledge, our method is much more sensitive than previous colorimetric reports for cysteine and histidine detection. The cost of the detection is quite low since the developed array is label free and enzyme free by using low-cost DNA and Cu2+. Moreover, the colorimetric detection operation is very simple without any further modifications. AUTHOR INFORMATION

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS


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This work was supported by the National Natural Science Foundation of China (No. 21190040), the State Key Instrument Developing Special Project of Ministry of Science and Technology of China (No. 2012YQ170003), the Instrument Developing Project of the Chinese Academy of Sciences (No. YZ201203) and the Natural Science Foundation of Jilin Province, China (No. 20130101117JC).

ASSOCIATED CONTENT

Additional information about the circular dichroism (CD) spectra of G-quadruplex and the Tables of LOD comparision for histidine and cysteine detected by different arrays. This material is available free of charge via the Internet at http://pubs.acs.org.

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FIGURE CAPTIONS

Scheme 1. The schematic illustration of the colorimetric strategy for histidine and cysteine detection. G-quadruplex-Cu(II) metalloenzyme assembled by G-quadruplex and Cu2+ catalyzes the TMB oxidation reaction and generates a coloric change from colorless to blue. Upon the addition of His or Cys, Cu2+ prefers to combine with His or Cys rather than with G-quadruplex, reducing the catalytic activity on the colorimetric reaction of TMB.



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Figure 1. (A) Absorption curves (a) no Cu2+ and (b) 2.0 uM Cu2+ in G-quadruplex and TMB-H2O2 system. (B) The absorption values at 452 nm (A452) upon different concentrations of Cu2+ (from bottom to up: 1.5, 1.75, 2.0, 2.25, 2.5, 3.0, 3.5 uM) in G-quadruplex and TMB-H2O2 system. [G-quadruplex] = 100 nM, [TMB] = 0.4 mM, [H2O2] = 100 mM.

Figure 2. Absorption curves (a) no histidine and (b) 10 uM histidine in the G-quadruplex-Cu(II) metalloenzyme sensing system. [Cu2+] =2.5 uM, [G-quadruplex] = 100 nM, [TMB] = 0.4 mM, [H2O2] = 100 mM.


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Figure 3. (A) The absorption spectra against different concentration of histidine (from top to bottom: 0 nM, 10 nM, 50 nM, 100 nM, 300 nM, 500 nM, 1 uM, 2.5 uM, 5 uM, 10 uM, 15 uM, 20 uM, 30 uM). (B) The corresponding plots ofΔA452 values upon different concentrations of histidine. Inset: linear relationship betweenΔA452 and histidine over the range of 10 nM - 1 uM. (C) The absorption spectra upon different concentration of cysteine (from top to bottom: 0 nM, 5 nM, 25 nM, 50 nM, 150 nM, 300 nM, 500 nM, 1 uM, 2.5 uM, 5 uM, 10 uM, 20 uM). (D) The corresponding plots ofΔA452 values with different concentrations of cysteine. Inset: linear relationship betweenΔA452 and cysteine over the range of 5 nM - 500 nM. ΔA452 =A0 – A, A0 is the absorption value at 452 nm upon no target, A is the absorption value at 452 nm upon different concentration of target. [Cu2+] =2.5 uM, [G-quadruplex] = 100 nM, [TMB] = 0.4 mM, [H2O2] = 100 mM. The error bars were obtained based on three independent experimental results.



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Figure 4. Absorption curves of (a) no target, (b) cysteine and (c) cysteine/NEM in the sensing system. [cysteine] = 20 uM, [NEM] = 2.5 mM, [Cu2+] =2.5 uM, [G-quadruplex] = 100 nM, [TMB] = 0.4 mM, [H2O2] = 100 mM.

Figure 5. The bars of absorption change values (∆A452) with 10 uM histidine and various animo acids (20 uM). NEM was added in cysteine for incubating 60 min to eliminate disturb of cysteine. [NEM] = 2.5 mM, [Cu2+] =2.5 uM, [G-quadruplex] = 100 nM, [TMB] = 0.4 mM, [H2O2] = 100 mM.


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For TOC only


Colorimetric Strategy for Highly Sensitive and Selective Simultaneous

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