Detection of radon based on the lead-induced conformational change in aptamer T30695

Abstract

A novel simple and rapid strategy has been developed for the highly selective detection of accumulated radiation from radon and its decay products based on the lead-induced formation of a G-quadruplex. This method explores a new field in biological analysis that exploits the potential use of aptamers as sensors for gases and radioactive substances.

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Radon is a radioactive noble gas commonly found in soils, water and some materials used in construction and interior decoration.^1,2 Radon enters the human body through inhalation and causes internal injuries by radiation when it decays. The World Health Organization has classified radon as one of 19 environmental carcinogens³ and the International Agency for Research on Cancer has confirmed that radon and its decay products are carcinogens and represent the second highest risk factor for lung cancer.^4,5 These warnings reflect the widespread concern about the threat of radon to human health. The detection of radon is therefore an important task.

The instruments currently used to measure radon and its decay products are based on the detection of alpha particles. The determination of radon can be divided into three categories based on the sampling method: instantaneous sampling, continuous sampling and cumulative sampling.^6,7 However, the traditional method of measuring the accumulated amount of radiation from radon is greatly affected by climatic factors, such as temperature and humidity, and the measurement procedures are both complicated and time consuming. Furthermore, the on-site sampling and measurement process exposes operational personnel to radiation. Even in a laboratory setting, it is highly hazardous to perform measurements involving radioactive materials. Over the years, scientists have developed many new methods of detecting radon.^8–10 Talha et al.¹¹ reported a novel approach to measuring radon in water in the field by inserting a MEDUSA gamma ray detector into a 210 or 1000 L container.¹¹ Simulations using the Monte Carlo computational method allow the simultaneous detection of ²²⁰Rn and its isotope ²²²Rn.^12,13 Although these methods overcome the shortcomings of many traditional techniques, they are limited to real-time monitoring and therefore cannot measure the cumulative radiation dose from radon. The most practical way to measure radon is based on cumulative radiation and its decay products because this results in the major damage that radon causes to the human body.^14,15

Many efforts have been made to develop sensitive and selective sensors for Pb²⁺ and sensors using G-quadruplexes as the sensing elements have been reported.^16–19 G-quadruplexes are secondary structures of DNA formed by guanine-rich oligonucleotides, which are prevalent in the human genome. Compared with other common metal ions, Pb²⁺ strongly induces the formation of G-quadruplexes as a result of the match between the cavity in G-quadruplexes and the ionic radius and charge of the Pb²⁺ ion. This provides a highly selective and sensitive strategy for the determination of Pb²⁺.

We propose here a simple and rapid strategy for the highly selective detection of accumulated radiation from radon and its decay products. We established a novel measuring system for radon based on lead-induced G-quadruplex formation. The whole process protects operating personnel from potential radiation hazards in a rapid and simple manner. Scheme 1 shows the sensing mechanism for the detection of radon. Radon has a very short half-life and its decay yields lead as a stable daughter product.²⁰ We collected the Pb²⁺ from decaying radon in acetic acid. We used a disposable plastic Petri dish as a sample container and added acetic acid as the absorbent. As the aerodynamic diameter of these radioactive aerosols is >1 μm, a mixed cellulose microporous membrane with a pore size of 0.8 μm was used to seal the top of the plate to prevent contaminants from entering.^21–23 The Petri dish was then exposed to a radon chamber for the passive sampling of accumulated radiation. The guanine-rich, single strand T30695 formed a double strand with its complementary strand, C-16. On reaction with the fluorescent reagent PG, the double strand produced a strong fluorescence (F₀).²⁴ Radon (²²²Rn) decayed to yield a stable daughter product, lead. The Pb²⁺ ions induced a conformational change in T30695 to create a stable G-quadruplex, thereby preventing it from forming a double strand with C-16.^25,26 Because the double strand was inhibited, the fluorescence (F) that occurred on the addition of PG became weaker. A higher concentration of radon yielded a higher concentration of Pb²⁺ ions, which caused a more substantial change in the fluorescence intensity. This formed the basis of a non-radioactive, label-free, fluorescent DNA sensor that could be used for the highly sensitive detection of radon based on the conformational change induced in the aptamer T30695 by lead ions.

Scheme 1 Schematic diagram of the label-free fluorescent detection of radon based on the lead-induced conformational change in T30695.

The results shown in Fig. 1 illustrate that the principle of this analysis was correct. The T30695 had a random coiling state without a base pairing plane. The fluorescence intensity of the T30695 + PG system was weak. When the complementary strand C-16 was added to the solution with the T30695, the system T30695 + C-16 + PG showed a significantly enhanced fluorescence. The two single strands formed a double helix structure and the positively charged PG was inserted into the double strand plane and the minor groove of the double helix, generating a strong fluorescence.²⁷ Lead resulting from the decay of radon dissolved in a 0.2% solution of acetic acid to form Pb²⁺ ions. On incubating a solution of Pb²⁺ ions and T30695, the Pb²⁺ induced T30695 to form a tight G-quadruplex structure, preventing it from forming a double helix structure with its complementary strand C-16. As PG cannot bind to the G-quadruplex structure, only a weak fluorescence was detected in the T30695 + Pb²⁺ + C-16 + PG system. Therefore, because a greater concentration of radon yielded a greater concentrations of Pb²⁺ ions, which, in turn, resulted in greater changes in the fluorescence intensity, the fluorescence intensity could be used as a sensor to measure the concentration of radon. The fluorescence intensity was not reduced to the same level as in the T30695 + PG system after the addition of Pb²⁺. This may be because a small amount of the double strands formed from the competitive binding of T30695 and C-16 resulted in a weak fluorescence. After T30695 had changed from a single strand to its quadruplex form, the spatial charge density increased on the DNA strand.^28,29 PG had a higher affinity for the G-quadruplex than for a single strand. Therefore the fluorescence intensity was slightly higher than that of PG with the single stranded T30695.

Fig. 1 Fluorescence emission spectra of PG in Tris–HAc (10 mM, pH 7.0) buffer solution under different conditions. The concentrations of T30695, C-16, Pb²⁺, Bi²⁺and PG were 10 nM, 14 nM, 60 nM, 60 nM and 1× , respectively.

It was difficult for interfering substances in the air to enter the sample dish under these sampling conditions because the opening of the sample dish was sealed with a microporous membrane. The process of radon decaying to lead produces polonium and bismuth as intermediate daughter products. As a result of the extremely short half-life of polonium (3.1 min) and the fact that it is one of the rarest elements, the polonium content in the samples was very low and had a negligible impact on the measurements. Therefore we focused on studying the potential interference from bismuth. Using the same experimental setup as in Fig. 1, we replaced lead with 60 nM bismuth. The detected fluorescence intensity was almost identical between the T30695 + Bi³⁺ + C-16 + PG and T30695 + C-16 + PG systems. Therefore we concluded that the presence of Bi³⁺ did not interfere with the results.

As certain heavy metal ions can suppress the fluorescence of fluorescent dyes,³⁰ we performed the experiment with a random oligonucleotide strand R, which had the same number of bases as T30695 (Fig. S4 ESI†). When a standard solution of 60 nM Pb²⁺ ions or a radon radiation sample solution was added, the F–λ curves of R + C–R + PG, R + Pb²⁺ + C–R + PG and R + sample + C–R + PG almost overlapped, the fluorescent intensity remained unchanged. These data suggest that the reduction in fluorescence intensity shown in Fig. 1 is not due to the inhibitory effect of Pb²⁺ on PG and the sample itself does not interfere with the detection of radon.

To better illustrate the reaction between Pb²⁺ and T30695, as well as the conformational changes in T30695, we conducted an experiment using circular dichroism spectrometry (Fig. 2). A positive peak appeared at 265 nm for single-chain T30695. The positive peak increased after adding Pb²⁺. Simultaneously, a negative peak appeared at 240 nm. These results show that Pb²⁺ induced the formation of a parallel G-quadruplex structure in T30695.^31,32 After incubating T30695 and C-16 alone, a positive peak appeared between 260 and 285 nm and a distinct negative peak appeared at 240 nm. This reflects the typical circular dichroism spectra of the B-type oligonucleotide double helix structure formed by polyguanine and cytosine.³³ After incubating T30695 and Pb²⁺ for a period of time, C-16 was added. The scanned circular dichroism spectrum produced by this solution was not very typical, however. A negative peak appeared at 240 nm and a large positive peak appeared at 285 nm with a shoulder at 265 nm. This is probably due to the coexistence of the single-chain T30695 structure, the G-quadruplex structure and the B-type double helix structure in the system, producing positive peaks at both 285 and 265 nm.

Fig. 2 Circular dichroism spectra of 5 μM T30695 and 7 μM C-16 in the presence and absence of 30 nM Pb²⁺.

Fig. 3 shows that, as the concentration of Pb²⁺ ions increased from 0 to 60 nM, the fluorescence intensity decreased. When the Pb²⁺ concentration exceeded 60 nM, the amplitude of the reduction in fluorescence intensity decreased. Fig. 3B shows that when the concentration of Pb²⁺ was maintained in the range 1–60 nM, a good linear relationship existed between the difference in fluorescence (ΔF) and the Pb²⁺ concentration, as described by the regression equation ΔF = 22.78c_Pb (nM) + 10.38 (r = 0.9955); the detection limit was 0.26 nM.

Fig. 3 (A) Fluorescence emission spectra of the sensing system on exposure to 0, 1, 2, 5, 8, 10, 20, 30, 40, 50, 60, 100, 150 and 200 nM Pb²⁺. (B) Calibration graph of the sensing system for Pb²⁺ (0–60 nM). Error bars represent standard deviations from three repeated measurements.

Radon samples with cumulative concentrations of 1.98, 4.46, 8.52, 12.43, 16.97, 22.04, 28.84, 35.44 and 42.84 (×10⁴ Bq h m⁻³) were collected and numbered from 1 to 9. Each sample was tested in parallel six times to determine the accuracy of the method. At the same time, we performed standard recovery tests to determine the accuracy of this method. Table 1 and Fig. 4 show that as the cumulative concentration of radon increased, the lead content of the sample and ΔF increased in proportion, showing a good logarithmic relationship between the two components in the range 1.98–42.84 (×10⁴ Bq h m⁻³). The regression equation was ΔF = 469.02 ln(c_Rn + 5.67) − 976.11 (r = 0.9926). However, in the range 1.98–22.04 (×10⁴ Bq h m⁻³), there was a good linear relationship between the two components and the regression equation was ΔF = 29.03c_Rn + 0.7734 (r = 0.9948). Therefore we established a mathematical quantitative model between the cumulative concentrations of radon and its daughter lead. The detection range for this method was 1.98–22.04 (×10⁴ Bq h m⁻³) with a detection limit of 1024 Bq h m⁻³.

Table 1 Detection of Pb²⁺ in samples

Sample	ΔF (a.u.)	Found^a (nmol L^−1)	Added^b (nmol L^−1)	Total^c found	RSD (%)	Recovery^d (%)
a Found represents the detection values of Pb^2+ in the sample.b Added represents the detection value of the Pb^2+ added to the sample.c Total found represents the total amount of Pb^2+ detected after Pb^2+ was added to the sample.d Recovery represents the ratio of [(total found − found)/added].
1	57.47	2.07	5	7.21	3.72	102.8
2	113.1	4.50	20	25.28	3.58	103.9
3	248.7	10.43	20	30.95	2.66	102.6
4	383.4	16.36	20	36.69	2.41	101.65
5	513.7	22.06	20	41.88	2.10	99.10
6	617.5	26.63	10	36.85	2.55	102.20
7	694.3	30.00	10	39.75	2.47	97.50
8	751.2	32.51	10	42.34	2.72	98.30
9	798.8	34.58	10	44.28	2.86	97.00

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Fig. 4 Relationship between the fluorescence intensity and the concentration of radon. The larger graph shows the fitted logarithmic regression of radon in the range 1.98–42.84 (×10⁴ Bq h m⁻³). The inset shows the fitted linear regression of radon in the range 1.98–22.04 (×10⁴ Bq h m⁻³).

We have developed a non-radioactive, label-free, fluorescent DNA sensor that can be used for the detection of radon. Radon quickly decays to lead. The use of the radiation characteristics of radon and sample collection with a cover meant that there was almost no interference from other chemicals and the analysis was low cost. The whole process protects operation personnel from potential radiation hazards in a rapid and simple manner. This is a novel approach to measure cumulative radon concentrations in the environment by using nucleic acid aptamers and explores a new field in biological analysis by exploiting the potential of aptamers as sensors for minerals, gases and radioactive substances.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (No. 81473021).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra03481k

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