A label-free biosensor for selective detection of DNA and Pb²⁺ based on a G-quadruplex

Abstract

A highly sensitive and selective label-free biosensor was designed for DNA determination based on target-induced opening of the hairpin DNA and forming a G-quadruplex with protoporphyrin IX (PPIX) as a signal reporter. In the absence of target-DNA (T-DNA), the probe-DNA (P-DNA) forms a hairpin structure. A G-rich sequence in the strand is partially caged in the stem-loop structure and cannot fold into a G-quadruplex. In the presence of T-DNA, the specific combination of targets with the sensing sequence (loop sequence) triggers the release of the G-rich sequence and allows it to fold properly and bind with PPIX resulting in the fluorescence of the G-quadruplex–PPIX providing the readout signal for the sensing event (detection limit of 3.5 nM). Based on the higher efficiency of Pb²⁺ at stabilizing G-quadruplexes than K⁺ and the Pb²⁺-stabilized G-quadruplexes not binding to PPIX, which resulted in fluorescence decrease, the proposed DNA-sensing system can be further exploited as a Pb²⁺-sensing method. The detection limit for Pb²⁺ is 2.6 nM.

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1. Introduction

As we all know, as a carrier of genetic information, DNA is of great importance in life activities. The sequence-specific detection of DNA plays a significant role in many areas, such as environmental monitoring,¹ analysis of food,² detecting crime,³ detection of genetic disorder⁴ and clinical diagnostics.⁵ Because of its huge potential application, it is highly desirable to develop ultrasensitive and specific methods for DNA detection. To meet these requirements, enormous efforts have been put into research and various kinds of methods have been developed for the detection of DNA such as electrochemistry,⁶ surface plasmon resonance,⁷ fluorescence resonance energy transfer (FRET),^8,9 strand displacement,¹⁰ phosphorescence energy transfer (PET),¹¹ and photoinduced electron transfer.¹² However, these reported detectors may have not only a high cost of operation but potentially complex processes. In recent years, the use of nicking endonuclease greatly promoted the development of signal amplification.^13,14 Unfortunately, this kind of enzyme requires target DNA with a specific recognition site, which limits the general applications of it. Unlike the nicking endonuclease, exonuclease III (Exo III)^15,16 is a sequence-independent enzyme which does not require a specific recognition site. In addition, Exo III can catalyze the stepwise removal of mononucleotides from 3′-hydroxyl termini of double-stranded DNA, which is not active on 3′-overhang ends of double-stranded DNA or single-stranded DNA. However, multi-step nucleases application would increase the assay cost, and the need for relatively complex design procedures limits its wide application. Recently, Willner et al. put forward a Mg²⁺-dependent DNAzyme as a biocatalyst to avoid the use of protein-based enzymes. But this does not get rid of the higher synthesis cost and the risk of instability coming from ribonuclease and chemical degradation. So, further studies are still essential to develop some convenient and economic methods of detection of DNA.

Lead ions (Pb²⁺) are one of the most toxic environmental pollutants in aquatic ecosystems and contaminants adversely affect the human health.^17,18 Thus the ultrasensitive and quantitative detection of Pb²⁺ is of considerable significance. On account of the complicated pretreatment procedures and the costly apparatus, the conventional methods such as atomic absorption spectroscopy (AAS)¹⁹ or inductively coupled plasma mass spectrometry (ICP-MS)²⁰ are limited to use for daily on-site and real-time analysis. In the past few years, functional nucleic acids became a powerful and convenient detection platform for Pb²⁺ analysis. Among them, the Pb²⁺ dependent RNA-cleaving DNAzymes (such as 8–17E DNAzyme and GR-5 DNAzyme) are the very common sensing elements for Pb²⁺ sensors.^21–26 However, many of these systems have limited practical use because of, for example, high cost (e.g., enzymes), complicated processing and the use of unstable molecules (e.g., RNA).²⁷ In addition, a Pb²⁺-induced allosteric G-quadruplex is also utilized for Pb²⁺ detection. For example, Dong's group introduced a Pb²⁺-induced allosteric G-quadruplex DNAzyme, which can serve as a Pb²⁺ sensor.²⁸ However, the need of hemin and H₂O₂ as cofactors might limit their application. So, the design of convenient and economical approaches is still in great demand for the sensitive and selective detection of Pb²⁺ with rapid and easy manipulation.

Protoporphyrin IX (PPIX) is an unsymmetrical anionic porphyrin. Generally, PPIX is inclined to aggregate into micelles with low fluorescence in aqueous solution, while its fluorescence intensity is enhanced via binding to parallel G-quadruplex.²⁹ Herein, we use PPIX as a parallel G-quadruplex-specific fluorescent probe for monitoring DNA structural changes and utilize it to develop a DNA and Pb²⁺ sensor. This assay is not only sensitive and reliable, but also simple and economical in operation.

2. Experimental

2.1 Materials and reagents

The oligonucleotide strands (Table 1) were purchased from Sangon Biotechnology Co. Ltd. (Shanghai, China) and purified by HPLC. Tri(hydroxymethyl)aminomethane (Tris) was bought from Sinopharm Group Co. Ltd. (Shanghai, China). Protoporphyrin IX (PPIX) was acquired from Aladdin Reagent Co. Ltd. (Shanghai, China). All other reagents are analytical grade and used without further purification. Doubly distilled water was used throughout the work.

Table 1 The oligonucleotides used in this work^a

a The bold letters identify stem portion, the underlined italic letters identify loop portion, and the underlined bold letters identify mismatched bases.
Probe DNA	5′-ACC CAC T̅T̅T̅ T̅T̅C̅ G̅T̅T̅ T̅C̅C̅ GTG GGT AGG GCG GGT TGG-3′
Target DNA	5′-GGA AAC GAA AAA-3′
Single-base mismatched DNA	5′-GGA AAG̅ GAA AAA-3′
Two-base mismatched DNA	5′-GGA AAG̅ GAA T̅AA-3′
Three-base mismatched DNA	5′-GC̅A AAG̅ GAA T̅AA-3′

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2.2 Preparation of probe

The oligonucleotides were prepared in the Tris–HCl buffer (50 mM Tris–HCl, pH = 7.4) and quantified using UV-vis absorption spectroscopy with the following extinction coefficients (ε₂₆₀ nm, M⁻¹ cm⁻¹) for each nucleotide: A = 15 400, G = 11 500, C = 7400, T = 8700. The probe-DNA (P-DNA) solutions were heated at 95 °C for 10 min and gradually cooled to room temperature. Then, freshly prepared PPIX solution and coordination cations were added into the DNA solution. So we got the probe solution.

2.3 Circular dichroism (CD) study

CD spectra were measured on a Applied Photophysics at 25 °C. The DNA solution (1 μM) was prepared in 50 mM Tris–HCl buffer (pH 7.4) containing 100 mM KCl, 10 mM MgCl₂. The mixture was heated to 95 °C for 10 min to remove aggregates, gradually cooled to 25 °C, and then incubated at 25 °C for 20 min. To this solution was added 1 μM T-DNA. Then the mixture was incubated at 25 °C for another 1 h. CD spectra were recorded between 240 and 320 nm with 1 cm path length cuvettes. Spectra were averaged from three scans, which were recorded at 100 nm min⁻¹ with a response time of 0.1 ms and a bandwidth of 1 nm.

2.4 Detection of DNA

The P-DNA solution (1 μM) was prepared in 50 mM Tris–HCl buffer (pH 7.4) containing 100 mM KCl and 10 mM MgCl₂. The mixture was heated to 95 °C for 10 min to remove aggregates, gradually cooled to 25 °C, and then incubated at 25 °C for 20 min. Then, to this solution was added different concentrations of T-DNA and PPIX (1 μM). The mixture was allowed to incubate at 25 °C for another 1 h. The fluorescence emission spectra of DNA–PPIX complexes in the Tris–HCl buffer were recorded from 550 nm to 750 nm by using a Cary Eclipse fluorescence spectrometer (Varian). The excitation wavelength was set at 410 nm.

2.5 Detection of Pb²⁺ ion

The P-DNA solution (1 μM) was prepared in 50 mM Tris–HCl buffer (pH 7.4) containing 100 mM KCl and 10 mM MgCl₂. The mixture was heated to 95 °C for 10 min to remove aggregates, gradually cooled to 25 °C, and then incubated at 25 °C for 20 min. Then, to this solution was added 1 μM T-DNA, 1 μM PPIX and different concentrations of Pb²⁺. The mixture was allowed to incubate at 25 °C for another 1 h. The fluorescence emission spectra of the reaction product in the Tris–HCl buffer were recorded from 550 nm to 750 nm by using a Cary Eclipse fluorescence spectrometer (Varian). The excitation wavelength was set at 410 nm.

3. Results and discussion

3.1 Principle of sensing DNA

It's reported that, PPIX exhibits a dramatic fluorescence enhancement upon binding to parallel G-quadruplexes. However, similar properties were not observed in the presence of duplex, triplex or single-stranded nucleic acid structure.²⁹ This allows us to utilize PPIX as a fluorescent reporter and monitor the conformational change.

Here we have designed a label-free hairpin structure of oligonucleotide probe, which mainly consists of three parts: stem portion, loop portion (specifically bind with T-DNA) and G-rich area (Scheme 1). In the absence of T-DNA, P-DNA forms hairpin structure. In the meanwhile, part of G-rich bases is caged in the stem-loop structure and thus G-rich bases cannot form into G-quadruplex structure, so there is no fluorescence signal. In the presence of T-DNA, the hairpin structure is open and releases the G-rich area to fold into G-quadruplex structure through hybridizing with T-DNA, thus forming G-quadruplex–PPIX complex with strong fluorescence signal which is closely related to T-DNA concentration.

Scheme 1 Schematic representation of the DNA and Pb²⁺ detection method.

As shown in Fig. 1, in the absence of T-DNA, the sensing system showed a very low fluorescence signal, which was slightly higher than a system containing PPIX only. This indicated that the G-rich sequence in the P-DNA strand could not fold into a G-quadruplex, and had almost no effect on PPIX except the emission wavelength blue-shifted from 623 nm to 634 nm. In the presence of 1 μM T-DNA, the fluorescence signal of the sensing system increased dramatically because of formation of G-quadruplex–PPIX complex, which was attributed to a T-DNA-mediated structural switch of P-DNA and the formation of a parallel G-quadruplex that strongly bound with PPIX.

Fig. 1 Fluorescence spectra of 50 mM Tris–HCl (pH 7.4, 100 mM K⁺, 10 mM Mg²⁺) containing 1 μM PPIX (curve 1), 1 μM PPIX + 1 μM P-DNA (curve 2), 1 μM PPIX + 1 μM P-DNA + 1 μM T-DNA (curve 3) at 25 °C.

3.2 CD measurements

In order to verify our speculation, we examined the structural changes in the DNA strand due to the addition of T-DNA using CD measurements. It has been reported that CD spectra of a typical parallel G-quadruplex structure have a positive peak near 260 nm and a negative peak around 240 nm, and that of a typical antiparallel G-quadruplex structure have a positive peak at 295 nm and a negative peak close to 265 nm.^30,31 P-DNA in buffer solution (50 mM Tris, pH 7.4, 100 mM KCl, 10 mM MgCl₂) displayed a positive peak around 276 nm confirming that there was no G-quadruplex in the absence of T-DNA (black curve in Fig. 2). However, the P-DNA/T-DNA mixture showed a positive peak at around 258 nm, which was a typical characteristic of a parallel G-quadruplex structure, indicating that the addition of T-DNA indeed can induce the formation of parallel G-quadruplex.

Fig. 2 The CD spectra of P-DNA (curve 1) and P-DNA + T-DNA (curve 2) and P-DNA + T-DNA + Pb²⁺ (curve 3) in 50 mM Tris–HCl (pH 7.4, 100 mM K⁺, 10 mM Mg²⁺) at 25 °C.

3.3 Optimization of sensing conditions

Fluorescence titration experiments were then performed to explore the optimal sensing conditions. All the measurements were performed three times, and the error bars represented for standard deviation (SD) across three repetitive experiments. As PPIX concentration was increased, we found that the fluorescence signal enhanced greatly and little change was observed when the concentration exceeded 1 μM (Fig. 3). So, the PPIX concentration of 1 μM was selected in subsequent experiments.

Fig. 3 Optimization of the concentration ratio of DNA/PPIX. Experiments were carried out in a 50 mM Tris–HCl (pH 7.4) buffer containing 1 μM P-DNA + 1 μM T-DNA, 100 mM K⁺, 10 mM Mg²⁺ at 25 °C.

We also found that K⁺ concentration had a great influence on the fluorescence signal. As shown in Fig. 4A, within certain scope of the K⁺ concentration, the signal increased with K⁺ concentration, and reached a maximum with 100 mM of K⁺. When the concentration of K⁺ exceeded 100 mM, the fluorescence signal decreased slightly with the addition of K⁺. In addition, we observed a similar phenomenon on the influence of the Mg²⁺ concentration, and the signal reached a maximum with 10 mM of Mg²⁺ (Fig. 4B). Therefore, to ensure a good signal-to-background ratio, we chose Tris–HCl buffer solution with 1 μM of PPIX, 100 mM of K⁺ and 10 mM of Mg²⁺ as reaction condition in the following experiments.

Fig. 4 (A) Influence of K⁺ on the detection system. (B) Influence of Mg²⁺ on the detection system. (C) Optimization of the reaction temperature. (D) Influence of pH on the detection system. Experiments were carried out in a 50 mM Tris–HCl (pH 7.4) buffer containing 1 μM P-DNA + 1 μM T-DNA at 25 °C. F₀ and F are the fluorescence intensities in the absence and presence of T-DNA respectively.

To investigate the influence of reaction temperature on the reaction system, experiments were then conducted in different temperature conditions (Fig. 4C). All the measurements were performed three times. The results showed the fluorescence signal was maximum at 25 °C. A further study was performed to investigate the fluorescence response of G-quadruplex–PPIX complexes on different pH values (as shown in Fig. 4D). The fluorescence response showed the pH of solution had a great influence on detection system. The fluorescence intensity increased from pH 6.0 and reached a maximum at pH 7.4, followed by a decrease to pH 9.0. Accordingly, a buffer solution of pH 7.4 was selected in subsequent experiments.

3.4 Sensitivity of the DNA-sensing system

To demonstrate the feasibility of our proposed approach, fluorescence signals for different concentrations of T-DNA were measured under the optimal conditions. All the measurements were repeated for three times. Fig. 5A illustrated that increasing concentrations of T-DNA in the range from 0 to 2 μM resulted in gradual fluorescence increase of the G-quadruplex–PPIX, which was caused by T-DNA hybridizing with P-DNA, then opening hairpin structure with G-rich liberation, and thus forming parallel G-quadruplex. From Fig. 5B, a linear relationship was observed over a range of 0–100 nM (R² = 0.9908) with a limit of detection of 3.5 nM (defined as a signal to noise ratio of 3). The sensitivity of the proposed method is similar or comparable to those of recently developed MBs (molecular beacons) or other label-free methods, for which nanomolar DNA targets can be efficiently detected.^32–36

Fig. 5 (A) Fluorescence spectra of 50 mM Tris–HCl (pH 7.4) containing 1 μM P-DNA in the presence of 0 nM to 2000 nM T-DNA under optimized reaction condition at 25 °C. (B) T-DNA concentration-dependent change in the fluorescence signal of the reaction solution. The excited wavelength was set at 410 nm. The inset shows the dependence of fluorescence intensity on T-DNA concentration.

3.5 Selectivity of the DNA-sensing system

To further validate the selectivity of this approach for DNA, different kinds of DNA including complementary DNA, single-base mismatched DNA, two-base mismatched DNA, and three-base mismatched DNA were chosen to investigate the selectivity of this approach. The fluorescence intensities at 634 nm for perfectly matched DNA, single-base mismatched DNA, two-base mismatched DNA, and three-base mismatched DNA were about 4.35, 3.14, 1.75, and 1.26 times higher than the background signal, respectively (Fig. 6). This showed the good selectivity of the proposed method. Therefore, this DNA sensing system could be used to discriminate perfectly matched and mismatched DNA, which should be attributed to the relatively long loop of the P-DNA with a thermodynamically stable hairpin structure. The high specificity of this method indicated great potential for single nucleotide polymorphism analysis.

Fig. 6 Fluorescence intensity for mismatched DNA and complementary DNA at 1 μM.

3.6 Principle of sensing Pb²⁺

Pb²⁺ has been reported to have higher efficiency than K⁺ at stabilizing G-quadruplexes, which indicates that Pb²⁺ could competitively bind to K⁺-stabilized G-quadruplexes, resulting in sharp changes in their structures and functions.^37–39 Here, we utilized this characteristic to sense Pb²⁺ by fluorescence technique. The basic principle of this biosensor for Pb²⁺ is shown in Scheme 1. In the absence of Pb²⁺, K⁺-stabilized G-quadruplex bound with PPIX, giving rise to high fluorescence. On addition of Pb²⁺, Pb²⁺ could gradually replace the position of K⁺ in the G-quadruplexes to form more compact DNA folds. Then PPIX were released from G-quadruplexes, which resulted in fluorescence decrease. The structure changes were also examined using CD measurement and the results were shown in Fig. 2.

To test the hypothesis, fluorescence spectroscopy was utilized to measure the fluorescence signal change of PPIX in different condition (Fig. 7A). The fluorescence of PPIX itself was very weak (curve 1 in Fig. 7A), but had a dramatic enhancement upon binding to K⁺-stabilized G-quadruplexes (curve 2 in Fig. 7A). On addition of Pb²⁺, the fluorescence decreased sharply (curve 3 in Fig. 7A) due to the formation of Pb²⁺-stabilized G-quadruplexes. This could suggested that the conformational change of G-quadruplexes resulted in the liberation of PPIX. A similar rule was also obtained by Guo's group that Pb²⁺ induced K⁺-stabilized G-quadruplex (PS2.M) to undergo a conformational change, which resulted in releasing NMM (N-methyl mesoporphyrin IX) from the G-quadruplexes, accompanied by a decrease in the fluorescence of K⁺-stabilized G-quadruplexes.³⁹ We also measured the fluorescence intensity of the detection system upon incubation with Pb²⁺ for different time intervals at room temperature. As displayed in Fig. 7B, the fluorescence intensity declined obviously with increasing time, and the curve reached a plateau within 30 min.

Fig. 7 (A) Fluorescence spectra of 50 mM Tris–HCl (pH 7.4, 100 mM K⁺, 10 mM Mg²⁺) containing 1 μM PPIX (curve 1), 1 μM PPIX + 1 μM P-DNA + T-DNA (curve 2), 1 μM PPIX + 1 μM P-DNA + 1 μM T-DNA + 2 μM Pb²⁺ (curve 3); (B) fluorescence intensity of G-quadruplex–PPIX–Pb²⁺ complexes vs. incubation time. Experiments were carried out in a 50 mM Tris–HCl (pH 7.4, 100 mM K⁺, 10 mM Mg²⁺) containing 1 μM PPIX + 1 μM P-DNA + 1 μM T-DNA + 2 μM Pb²⁺.

3.7 Sensitivity and selectivity of the Pb²⁺-sensing system

The feasibility of this method for Pb²⁺ quantitative detection was investigated. As shown in Fig. 8A, in the presence of 1 μM T-DNA, the sensing system showed a much high fluorescence signal. With the addition of different concentrations of Pb²⁺, the signal decreased gradually and reached a plateau when Pb²⁺ was close to 2 μM. A good linear response (R² = 0.9975) toward Pb²⁺ was seen at Pb²⁺ concentrations from 0 to 0.1 μM (the inset in Fig. 8B) suggesting that the Pb²⁺-sensing system can be used to quantify Pb²⁺. The limit of detection for Pb²⁺ was estimated to be 2.6 nM (S/N = 3), which was comparable to other DNA-based fluorescence sensors for Pb²⁺.^27,39–42

Fig. 8 (A) Fluorescence spectra recorded under optimized reaction condition in the presence of 0 nM to 2000 nM Pb²⁺. (B) Pb²⁺ concentration-dependent change in the fluorescence signal of the reaction solution. The inset showed the dependence of fluorescence intensity on Pb²⁺ concentration.

The selectivity of this biosensor was evaluated by testing the fluorescence emission ratio (ΔF/F₀) in the presence of individual metal ion. As shown in Fig. 9, although the concentration of other tested ion was larger than that of Pb²⁺, only Pb²⁺ caused a considerable large fluorescence emission ratio. Selectivity studies showed that this method was highly selective for Pb²⁺, and the presence of other metal ions did not interfere with Pb²⁺ detection.

Fig. 9 Selectivity of the detection system. The fluorescence emission ratio (ΔF/F₀) in the presence of individual metal ion (500 nM Pb²⁺, 3 μM Hg²⁺ and other metal ions are 5 μM). F₀ and F are the fluorescence intensities in the absence and presence of Pb²⁺ respectively.

3.8 Application

The feasibility of this method for Pb²⁺ ion quantitation in real samples was also tested. Using a standard addition method, Pb²⁺ was added to real samples to a final concentration of 70 nM, and recovery values were obtained from fluorescence changes in the calibration graph. The results were listed in Table 2. The recovery of added Pb²⁺ was 100.7–106.3%, indicating that this method could detect Pb²⁺ in real samples with little interference.

Table 2 The concentration of Pb²⁺ in water samples detected using the proposed biosensor

Samples	Pb^2+ (nM)		Recovery (%)
	Added	Recovered
a Mean values of three determinations.b Standard deviation.
Tap water	70.0	74.4^a ± 1.6^b	106.3
Purified water	70.0	70.5^a ± 2.4^b	100.7
Mineral water	70.0	72.6^a ± 0.6^b	103.7

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4. Conclusions

In summary, we have developed a simple approach for fluorescence detection of DNA with high sensitivity and selectivity by using only one unlabeled oligonucleotide. The addition of target DNA led to a structural switch in the oligonucleotide, accompanied by the formation of fluorescent G-quadruplex–PPIX complexes. This sensor enabled the selective measurement of DNA with a detection limit of 3.5 nM. Through changing the sensing sequence (loop sequence) as needed, the detection of different DNA sequences can be achieved. This simple, cost-effective DNA detection technique was further explored for highly sensitive and selective Pb²⁺ detection with the detection limit of 2.6 nM. The assay could be accomplished by using a common fluorophotometer and no sophisticated experimental technique, cofactors or any chemical modification of DNA was required, which offered the advantages of simplicity in design and in operation and cost efficiency from an application standpoint.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (21173071, 30970696), Key Project of Henan Ministry of Education (14A150018) and Key Programs of Henan for Science and Technology Development (142102310273).

Notes and references

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