Highly sensitive and portable mercury(II) ion sensor using personal glucose meter

Abstract

In this paper, a highly sensitive and portable mercury(II) ion sensor based on personal glucose meter (PGM) recording is proposed. Thymine–thymine mismatches in the capture and detection of DNA were used to recognize target Hg²⁺. Magnetic separation and hydrolysis of sucrose into glucose of DNA-invertase conjugation were employed to obtain the PGM signal. There was a linear relationship between the PGM signal and the concentration of Hg²⁺ in the range of 8.0 nM to 1 μM. A correlation coefficient of 0.995 was obtained and the relative standard deviation was 3.6% for a concentration of 100 nM Hg²⁺ (n = 9). The selectivity and performance of the Hg²⁺ sensor in lake water, tap water and river water were also studied, which suggested our method has a great potential to be used in real applications.

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1. Introduction

Mercuric ion (Hg²⁺) is a widespread heavy metal ion which may cause deleterious effects on human health and the environment.¹ A very low concentration of Hg²⁺ can lead to adverse human health effects. Water-soluble Hg²⁺ is one of the most stable forms of mercury pollution.^2,3 To prevent potential human exposure to Hg²⁺, it is of great significance to be able to selectively and sensitively detect water-soluble Hg²⁺. Great efforts have been made to develop sensitive and selective Hg²⁺ detection methods. Among those methods, some are constructed by using thymine (T)-containing oligonucleotides as the sensing elements.^4,5 As reported in previous papers, Hg²⁺ can bind to two T residues of DNA, with high selectivity and affinity, which provides a powerful tool to develop Hg²⁺ detection methods. Many Hg²⁺ detection methods based on traditional analytical techniques, such as atomic absorption spectrometry, atomic fluorescence spectrometry, mass spectrometry, inductively coupled plasma atomic emission spectrometry and cyclic voltammetry, have been developed.^6,7 Although high sensitivity and selectivity have been achieved, most of these analytical techniques are expensive, time-consuming and require sophisticated instrumentation.^8–12 This has limited their wider applications, such as on-site applications. Therefore, it is interesting and of significance to develop a highly sensitive, inexpensive, simple and point-of-use method to detect Hg²⁺.

Due to its low cost, simple operation and portability, the personal glucose meter (PGM) has attracted worldwide attention.^13,14 It is available in stores at a low price (as low as $10 for a meter), and has been integrated into cell phones for point-of-use application. PGMs are mainly used to monitor the glucose concentration in diabetic patients. In 2011, Yi Lu linked PGM with functional DNA sensors to achieve portable, low-cost and quantitative detection of targets other than glucose.¹⁵ This has provided an excellent alternative to develop sensitive, inexpensive, simple and point-of-use Hg²⁺ sensors. However, it is an invasive DNA approach toward targets, which sacrifices the sensitivity of detection.

In this work, we developed a highly sensitive and portable mercury(II) ion sensor using a PGM. As shown in Fig. 1, in the presence of Hg²⁺, the Hg²⁺ and detection DNA were captured and concentrated onto streptavidin-MNBs through T–Hg–T linkage and magnetic separation, respectively. After the washing away of unbound Hg²⁺ and detection DNA, the bound DNA-invertase can be used to catalyze the hydrolysis of sucrose into glucose with millions of turnovers, which transformed the concentration of Hg²⁺ into the level of glucose for monitoring by a PGM.

Fig. 1 Schematic illustration of the design principle of Hg²⁺ sensor using a PGM.

2. Experimental

2.1 Reagents and chemicals

Streptavidin-MNBs (350 nm in diameter, aqueous suspension containing 0.05% Tween-20, 0.1% bovine serum albumin (BSA) and 10 μM EDTA at a concentration of 3.324 × 10¹¹ beads mL⁻¹) were obtained from Bangs Laboratories Inc. (Fishers, IN). All oligonucleotides were synthesized and purified by Sangon (Shanghai, China). The sequences were as follows:

Capture DNA: 5′-biotin-AAAAAAAAAT̲T̲T̲CCGT̲T̲T̲CGCT̲T̲T̲-3′

Detection DNA: 5′-HS-AAAAAAAAAT̲T̲T̲GCGT̲T̲T̲GCCT̲T̲T̲-3′

All DNA oligonucleotides were diluted by TE buffer (10 mM Tris–HCl and 1.0 mM Na₂EDTA, pH 8.0). They were denatured at 95 °C for 5 min and naturally cooled down to room temperature before use. Grade VII invertase obtained from baker's yeast (S. cerevisiae), Tween-20, sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and BSA were purchased from Sigma (St. Louis, MO). Other chemicals were of analytical grade and obtained from standard reagent suppliers and used directly. All solutions were prepared with Milli-Q water (resistivity = 18 MΩ cm) from a Millipore system.

2.2 DNA-invertase conjugation

Firstly, amounts of 30 μL of detection DNA (1 mM), 2 μL of sodium phosphate buffer (1 M, pH 5.5) and 2 μL of TCEP solution (30 mM) were mixed, which was then incubated for 1 h at room temperature. The mixture was purified by Amicon-10K 10 times using buffer A (0.1 M NaCl, 0.05% Tween-20, 0.1 M sodium phosphate buffer, pH 7.3). Then, in order to conduct invertase conjugation, 1 mg of sulfo-SMCC was added into 400 μL of invertase solution (dissolved in buffer A), which was incubated for 1 h on a roller. The obtained mixture was purified through centrifugation and Amicon-100K using buffer A 10 times. Finally, the obtained sulfo-SMCC-activated invertase was mixed with the detection DNA, which was incubated for 48 h at room temperature. The mixture was purified by Amicon-100K 10 times using buffer A.

2.3 Procedures for Hg²⁺ detection using PGM

Firstly, 5 μL of streptavidin-MNBs was washed three times by using buffer A to remove the surfactants. Then, 50 μL of buffer A was added into the streptavidin-MNBs suspension. One microliter of this streptavidin-MNBs suspension was added into a tube and 10 μL of capture DNA was added to bind the streptavidin-MNBs, which was placed on a roller for 30 min. It was washed using buffer A 5 times and the streptavidin-MNBs were separated by a magnet. Then, 100 μL of Hg²⁺ at different concentrations were mixed with the obtained streptavidin-MNBs and 100 μL of DNA-invertase conjugate (obtained as described in Section 2.2). It was allowed to incubate for 2 h on a roller. The mixture was further washed 5 times using buffer A containing BSA (2 mg mL⁻¹). It was washed 5 times using buffer A. Finally, 100 μL of sucrose in buffer A (0.5 M) was added into the system and allowed to incubate for 3 h. An amount of 5 μL of the obtained mixture was detected by a PGM.

3. Results and discussion

3.1 Design principle of Hg²⁺ sensor using a PGM

The design principle of the Hg²⁺ sensor is schematically described in Fig. 1. The capture DNA was immobilized onto streptavidin-MNBs through the reaction between streptavidin and biotin. The detection DNA was modified with DNA-invertase conjugation. In this case, the detection DNA and capture DNA were separated, as the mismatch of T–T. In the presence of Hg²⁺, the detection DNA and capture DNA stitched together after formation of T–Hg–T complex. The Hg²⁺ and detection DNA were captured and concentrated onto the streptavidin-MNBs through T–Hg–T linkage and magnetic separation, respectively. Therefore, only in the presence of Hg²⁺ can the detection DNA modified with DNA-invertase conjugation be immobilized onto streptavidin-MNBs. We assumed the concentration of Hg²⁺ was proportional to the amount of DNA-invertase bound to the streptavidin-MNBs. After the washing away of unbound Hg²⁺ and detection DNA, the bound DNA-invertase can be used to catalyze the hydrolysis of sucrose into glucose with millions of turnovers, which transformed the concentration of Hg²⁺ into the level of glucose for monitoring by a PGM.

3.2 Optimization of experimental parameters

Optimization of the concentration of detection DNA. The concentration of detection DNA is essential to the performance of the Hg²⁺ sensor, as a shortage of detection DNA leads to an abundance of unbound Hg²⁺ and an excess of detection DNA leads to an increase of the background signal. Therefore, different amounts of DNA-invertase conjugation (5, 10, 50, 100, 200 and 300 μL) were used for the detection of Hg²⁺. The PGM signals for different amounts of DNA-invertase conjugation are shown in Fig. 2. The PGM signal increased with increasing amount of DNA-invertase conjugation until 100 μL and there was a peak at the amount of 100 μL. As the amount of DNA-invertase conjugation increased above 100 μL, there was a slight decline in the signal, which was caused by the increase of the background signal. Therefore, an amount of 100 μL of DNA-invertase conjugation was selected for the following study.

Fig. 2 Relationship between the amount of detection DNA and signal. Condition: each data point represents an average of 3 measurements (each error bar indicates the standard deviation).

Optimization of incubation time for Hg²⁺ and detection DNA. The performance of the Hg²⁺ sensor was strongly affected by the incubation time of Hg²⁺ and detection DNA. As shown in Fig. 3, the PGM signal increased gradually with increasing incubation time (in the presence of 100, 300 and 500 nM Hg²⁺) at the early stage and a maximum was obtained at 100 min. In order to obtain the best signal-to-background level, 100 min was selected as the incubation time for subsequent detection.

Fig. 3 Relationship between the incubation time and signal. The concentration of Hg²⁺ from top to bottom is 500, 300 and 10 nM.

3.3 Sensing performance for Hg²⁺ detection

In order to evaluate the performance of the Hg²⁺ sensor, we challenged it with a series of concentrations of Hg²⁺, covering a range of nearly 3 orders of magnitude (8.0 nM to 1 μM). Improved signal of PGM was observed with the increase of Hg²⁺ concentration and intensity of signal increased monotonically (nearly linearly) with the concentration of Hg²⁺ (as shown in Fig. 4). There was a linear relationship between the signal of the PGM and the concentration of Hg²⁺ in the range of 8.0 nM to 1 μM. A correlation coefficient of 0.995 was obtained and the relative standard deviation (RSD) was 3.6% for a concentration of 100 nM Hg²⁺ (n = 9), which was comparable to or even better than some other reported methods. Such an attractive detection limit of our sensing strategy can be primarily attributed to the enrichment effect of streptavidin-MNBs and the million turnovers of sucrose hydrolysis into glucose. Substantial Hg²⁺ and detection DNA in solution were collected onto the surface of the PGM and one sucrose on the detection DNA turns into millions of glucose for monitoring by the PGM. According to the maximum contamination level of Hg²⁺ in drinking water issued by the United States Environmental Protection Agency (EPA), our Hg²⁺ sensor has great application prospects for the point-of-use and routine monitoring of Hg²⁺ with a wide dynamic range and superior detection sensitivity. A comparison of the detection performance with traditional methods using different equipment for determination of Hg²⁺ is given in Table 1.

Fig. 4 Relationship between the concentration of Hg²⁺ and signal. Condition: each data point represents an average of 3 measurements (each error bar indicates the standard deviation).

Table 1 Comparison of the detection performance with traditional methods using different equipment for determination of Hg²⁺

Equipment	Method	Linear range (10^−7 mol L^−1)	Reference
Spectrofluorimeter	QDs/DNA/Au NPs based nanosensor	0.02–0.6	Anal. Chem., 83, 7061–7065
Test paper detection	Variation of color upon binding with reagent	100–4000	RSC Adv., 2012, 2, 3714–3721
UV-visible spectrophotometer	Coordination of Hg^2+ to gold nanoparticles	0.1–5	Analyst, 2011, 136, 1690–1696
Electrochemical workstation	Modification of glassy carbon electrode	0.01–1	Anal. Methods, 2014, 6, 4988–4990
Renishaw 2000	Interaction between silver nanoparticles and Hg^2+	90.9 pM detected	Nanoscale, 2012, 4, 5902–5905
Personal glucose meter	T–T mismatches recognize target Hg^2+	0.08–10	This work

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3.4 Selectivity

In order to evaluate the selectivity of the Hg²⁺ sensor for Hg²⁺, we challenged the Hg²⁺ sensor with a series of environmentally relevant metal ions, including Cd²⁺, Pb²⁺, Mn²⁺, Fe³⁺, Cu²⁺, Co²⁺, Ni²⁺, Al³⁺, Ca²⁺, Cr³⁺, Sn²⁺, Zn²⁺, and Ag⁺, using the same experimental procedures as those for Hg²⁺. These metal ions were selected as they may coexist with Hg²⁺ in drinking water. The response of the Hg²⁺ sensor to these metal ions is shown in Fig. 5. We found that the presence of those metal ions at 1 μM elicits negligible responses compared with that of 300 nM of Hg²⁺ and their mixture. The signal of PGM was almost equal to the background signal. These observations suggested that binding of Hg²⁺ relies on specific recognition and that capture DNA and detection DNA were highly specific for the capturing of Hg²⁺. What is more, the presence of abundant metal ions in the solution did not affect the detection of Hg²⁺, which suggests that our sensor holds great promise as a powerful tool to be applied for detection of Hg²⁺ in real samples.

Fig. 5 The response of Hg²⁺ sensor to different metal ions. The metal ions from left to right are Cd²⁺, Pb²⁺, Mn²⁺, Fe³⁺, Cu²⁺, Co²⁺, Ni²⁺, Al³⁺, Ca²⁺, Cr³⁺, Sn²⁺, Zn²⁺, Hg²⁺ and a mixture of the metal ions listed.

3.5 Real sample analysis

In order to evaluate the practical application of the proposed Hg²⁺ sensor, we challenged the Hg²⁺ sensor with a series of environmental water samples, namely lake water, tap water and river water, using the same experimental procedures as those for Hg²⁺ detection in buffer solution. The results of Hg²⁺ detection for the different water samples suggested that the signals of PGM in lake water, tap water and river water were similar to that in buffer solution. Furthermore, the Hg²⁺ sensor was challenged with different amounts of Hg²⁺ in different water samples for recovery tests. The results are summarized in Table 2. Satisfactory values of between 94 and 106% were obtained for the recovery experiments, which indicated that the possible interference from the different background composition of water samples on the Hg²⁺ sensor was negligible. The above results demonstrate that our introduced Hg²⁺ sensor can be successfully applied to Hg²⁺ analysis in real environmental samples.

Table 2 Determination of Hg²⁺ in environmental water samples^a

Sample	Added (nM)	Found (nM)	Recovery (%)
a Note: each sample was analyzed using our proposed sensor, and all values were obtained as an average of three repeat determinations ± standard deviation (mean ± SD).
Tap water	20	18.9 ± 4.6	94.5
	100	103.2 ± 7.9	103.2
	300	285.0 ± 9.4	95.0
Lake water	20	19.1 ± 4.9	95.5
	100	106.1 ± 8.1	106.1
	300	282.3 ± 9.2	94.1
River water	20	19.2 ± 5.1	96.0
	100	98.5	98.5
	300	287.9	96.0

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4. Conclusion

In summary, a highly sensitive and portable Hg²⁺ sensor was developed based on thymine (T)-containing oligonucleotide recognition and PGM recording. The Hg²⁺ was captured and concentrated on streptavidin-MNBs and the DNA-invertase conjugation on the detection DNA catalyzed the hydrolysis of sucrose into glucose with millions of turnovers, which transformed the concentration of Hg²⁺ into the level of glucose for monitoring by the PGM. There was a linear relationship between the PGM signal and the concentration of Hg²⁺ in the range of 8.0 nM to 1 μM. A correlation coefficient of 0.995 was obtained and the relative standard deviation (RSD) was 3.6% for a concentration of 100 nM Hg²⁺ (n = 9). What is more, the Hg²⁺ sensor has a high selectivity in buffer solution and a comparable performance in lake water, tap water and river water, which suggests that our proposed Hg²⁺ sensor has a great positional to be used in real applications.

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