Label-Free and Enzyme-Free Colorimetric Detection of Pb2+ Based

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Label-free and Enzyme-free Colorimetric Detection of Pb2+ Based on RNA-Cleavage and Annealing-Accelerated Hybridization Chain Reaction Zhijun Huang, Junman Chen, Zewei Luo, Xiaqing Wang, and Yixiang Duan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00410 • Publication Date (Web): 05 Mar 2019 Downloaded from http://pubs.acs.org on March 5, 2019

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Analytical Chemistry

Label-free and Enzyme-free Colorimetric Detection of Pb2+ Based on RNA-Cleavage and Annealing-Accelerated Hybridization Chain Reaction Zhijun Huang, Junman Chen, Zewei Luo, Xiaqing Wang, and Yixiang Duan* Research Center of Analytical Instrumentation, Key Laboratory of Bio-resource and Eco-environment, Ministry of Education, College of Life Science, Sichuan University, Chengdu P.R. China, 610065. ABSTRACT: A label-free and enzyme-free colorimetric sensor for rapid detection of Pb2+ is reported, which is based on the strategy of DNAzyme mediated RNA-cleavage combined with annealing-accelerated DNA hybridization chain reaction (HCR). As a trigger DNA, the substrate strand (STM) of DNAzyme can initiate HCR effectively. However, when it is cleaved by DNAzyme in the presence of Pb2+, the separation of DNA functional domains leads to serious decline in HCR efficiency. As a result, the difference in Pb2+ concentration converts into the difference of DNA assembly, which eventually leads to the color change of colloidal gold nanoparticles (AuNPs). In this work, a DNA strand (cGR5) completely complementary to the catalytic strand (GR5) of DNAzyme is used to improve the dissociation of STM to enhance the HCR efficiency. In addition, the simple operation of DNA annealing is first used to accelerate the HCR process, enabling the Pb2+ detection to be completed in about 30 min. As advantages of high sensitivity, good selectivity, strong anti-interference ability and good practical performance are achieved, it is anticipated that the cheap and simple colorimetric sensor will be helpful for on-site detection of environmental and food samples.

Heavy metal pollution is one of the most serious concerns that endanger human health and ecological system.1-3 Due to the properties of bio-accumulative and non-biodegradable,4-7 lead is one of the most toxic heavy metal ion that threaten human health, which can cause serious development disorders and mental illness.8-11 In addition, lead pollution is also a common phenomenon due to extensive industrialization or outdated water supply systems.6, 10, 11 Therefore, the routine detection of Pb2+ is critical for environmental monitoring and food safety assessment. Recently, many analytical techniques have achieved sensitive detection of Pb2+, and the techniques such as atomic absorption/emission spectroscopy,12-15 atomic fluorescence spectroscopy,16, 17 X-ray fluorescence spectroscopy18, 19 and inductively coupled plasma mass spectrometry20, 21 can achieve extremely high sensitivity. However, all these analytical methods require expensive equipment, complicated and time-consuming pretreatment and highly skilled operators, which greatly limit the wide application of these techniques and increase the difficulty of Pb2+ detection. In contrast, using simple biosensors to detect Pb2+ is a simpler and cheaperchoice, which can greatly facilitate the routine inspection.22, 23 Due to Pb2+ can strongly and specifically stabilize the G-quadruplex structure; many simple biosensors have been reported based on the highly specific interaction between Pb2+ and G-quadruplex.24-27 However, because the G-quadruplex DNA can also be stabilized by other metal ions like Na+, K+, Tl+, Sr2+ and Ba2+, the constructed biosensors always encounter more interference.22 In addition to G-quadruplex DNA, DNAzyme is another

kind of highly specific recognition element. And the DNAzyme mediated RNA-cleavage is the one of the most commonly applied strategies in biosensor construction.28, 29

The DNAzyme GR5, which initially selected by Breaker and Joyce, is most commonly used for Pb2+ detection due to its high catalytic activity and good metal selectivity.30 Based on the application of DNAzyme GR5, a variety of sensors, including fluorescence sensor,31-34 electrochemical sensor,5 quartz crystal microbalance sensor,35 as well as colorimetric sensor36, 37 were constructed to detect Pb2+. Among these sensors, the colorimetric sensor has the least dependence on the detection device because the generated signal can be directly identified by the naked eye. Therefore, colorimetric sensor is considered to be more suitable for real-time and on-site detection. Salt-induced gold nanoparticles (AuNPs) aggregation is a highly sensitive strategy for colorimetric signal generation. Since DNAzyme GR5 can catalyze the RNAcleavage in the presence of Pb2+ and split the substrate strand into short DNA fragments, these DNA conformational change can also cause a color change of AuNPs.38, 39 However, due to the RNA-cleavage can only produce limited DNA conformational changes, the color change generated by these reported sensors are not so obvious,38, 39 which is not conducive to reading the results directly through the naked eye. To overcome this shortcoming, hybridization chain reaction (HCR) is used to increase the change of DNA conformation and enhance the chromatic aberration of the colorimetric results. HCR is an enzyme-free and effective

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signal amplification strategy that can assemble the DNA hairpins into long dsDNA products. Due to DNA hairpins have more single-stranded domains, and each trigger DNA can assemble dozens of DNA hairpins, 40-45 enormous changes in DNA conformation can be generated with the help of HCR. In this work, it is found that the DNA strand (cGR5), which is completely complementary to the catalytic strand (GR5), can effectively promote the separation of substrate strand (STM) and improve the assembly efficiency of HCR. And a simple operation of DNA annealing is also applied to speed up the HCR and save the time of Pb2+ detection. As a result, with the help of cGR5 and annealing accelerated HCR, Pb2+ detection can be completed in about 30 min, and the significant color change can be indentified directly by the naked eye. Moreover, without the need of AuNPs modification, the colorimetric sensor is low-cost and easy-to-operate. As the proposed colorimetric sensor has achieved good selectivity, can resist the interference from other metal ions and obtain good performance in the detection of actual water samples, it is believed that this proposed simple colorimetric can facilitate the work of daily detection.

EXPERIMENTAL SECTION AuNPs preparation. AuNPs (12.28 ± 0.93 nm) were prepared by sodium citrate reduction of HAuCl4 according to the reported procedure.46, 47 In brief, 100 mL of 1 mM HAuCl4 was boiled and reflux with vigorous stirring, and 2.4 mL of 5% sodium citrate was rapidly added into the boiling solution, the mixed solution was kept boiling for 30 min and then cooled to room temperature. The resultant AuNPs were stored in dark at 4 ℃ for further use. The procedure of Pb2+ detection. The procedure of Pb2+ detection was simple, only mixing, incubation, annealing and UV-vis measurement were needed. In the typical Pb2+ detection procedure, solution A (the mixed solution of 10 μL of 1M NaAc, 10 μL of 100 mM Tris-HAc (pH 7.5), 2.5 μL of 1 μM GR5, 2.5 μL of 1 μM STM, and 59 μL of H2O) was kept at 95 ℃ for 5 min, and annealed to 25 ℃ with a rate of 0.1 ℃/s. Then, 2.5 μL of Pb2+ stock solution was mixed with 84 μL of solution A and incubated at 37 ℃ for 10 min. Subsequently, 13.5 μL of solution B (the mixed solution of 1 μL of 100 mM EDTA, 2.5 μL of 1 μM cGR5, 5 μL of 10 μM HA8 and 5 μL of 10 μM HB8) was added into the above mixture to achieve a total volume of 100 μL. (Although only a small volume of DNA mixture was required in colorimetric detection, in order to reduce the errors, we used a total volume of 100 μL all through the work.) The 100 μL mixture was annealed from 95 ℃ to 25 ℃ with a rate of 0.1 ℃/s. (Note that DNA annealing can be accomplished by slowly cooling down with boiling water without precise temperature control equipment as in Figure 2, but we used the SimpliAmp Thermal Cycler (ABI, USA) for incubation and annealing (about 12 min) in this study to improve the uniformity and shorten the detection time.) After annealing, 7.5 μL of the reaction mixture was mixed with 100 μL of AuNPs colloid. Followed by addition of 5.5 μL of 100 mM Tris-HAc (pH 7.5) and waited for 10 min, the AuNPs colloid was tested by Lambda 25 UV-vis spectrophotometer using the quartz cell with 10 mm path lengths at room

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temperature. (It was found that better colorimetric performance was achieved by using TrisHAc instead of NaCl to adjust AuNPs aggregation, including more obvious UV-vis absorption peak of aggregated AuNPs, more significant color change, and higher sensitivity. Although the mechanism was not clear and still under study, TrisHAc was used to adjust AuNPs aggregation to obtain better Pb2+ detection performance)

RESULTS AND DISCUSSION Working principle of the colorimetric sensor. The principle of the Pb2+ colorimetric sensor is shown in Scheme 1. The typical Pb2+ detection process can be divided into three stages, namely, target recognition stage, HCR signal amplification stage and visualization signal generation stage. Scheme 1 Schematic illustration of the Pb2+ colorimetric sensor based on DNAzyme mediated RNA-cleavage and annealing accelerated HCR. The cGR5 is completely complementary to GR5, which can hybridize with GR5 and help the detachment of split or unsplit STM. Annealing is used to accelerate the HCR process, and salt-induced

aggregation of unmodified AuNPs is used to generate visualization signals. In the first stage, the substrate strand (STM) can be split into two short fragments (ST and SM) at the ribonucleotide site by GR5 in the presence of Pb2+. The changes in DNA conformation caused by DNA fragmentation can lead to difference in AuNPs aggregation and resulting in the color variation.38, 39 However, as mentioned above, due to only limited changes in DNA conformation can be generated by the RNA-cleavage reaction, this simple strategy always provides weak color change and limited sensitivity. In the second stage, HCR is applied for signal amplification, which can strongly enhance the color change and improve the sensitivity of the colorimetric sensor. It is well known that HCR can be efficiently initiated in the presence of trigger DNA.40, 48-54

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Conversely, HCR is strongly inhibited once the toehold domain and the DNA migration domain of trigger DNA are separated.55, 56 In this work, STM is used as trigger DNA to initiate the assembly of DNA hairpins. Moreover, the DNA strand (cGR5) which complementary to GR5 can promote the dissociation of STM and leading to the improvement of HCR efficiency. As a result, by using DNAzyme mediated RNA-cleavage for target identification and using HCR for signal amplification, the detection of Pb2+ is converted into detection of HCR products. In the third stage, the unmodified AuNPs are used to detect the HCR products. The difference of DNA conformation between ssDNA and dsDNA results in their varied ability to stabilize AuNPs. The flexible ssDNA can be easily adsorbed onto the surface of AuNPs to enhance electrostatic repulsion, while rigid dsDNA is difficult to bend and combine with AuNPs. Therefore, ssDNA can better protect AuNPs from salt-induced aggregation.39 It is known that HCR products are long dsDNA chains and with few single-stranded domains, while the loop domain and the toehold domain of the substrate DNA hairpins are in the single-stranded form.40 In addition, since each trigger DNA can assemble a large number of DNA hairpins, the occurrence of HCR will lead to significant conformational transition from ssDNA to dsDNA. Therefore, HCR is an ideal and powerful signal amplification strategy in constructing salt-induced AuNPs aggregation based colorimetric sensors.57 In this work, STM is used as the trigger DNA to initiate HCR. In the absence of Pb2+, STM is not cleaved and can initiate HCR efficiently. Therefore, the HCR substrate DNA hairpins are assembled to be long dsDNA chains, and can no longer stabilize the AuNPs against salt-induced aggregation. By contrary, in the presence of target ion Pb2+, STM is fragmented into split toehold domain (ST) and the DNA migration domain (SM), which can no longer trigger HCR efficiently. As a result, with the increase of Pb2+ concentration, the aggregation of AuNPs decreases, and the color of AuNPs colloid changes from blue to red, which can be easily observed by naked eye. Feasibility verification of the Pb2+ colorimetric sensor. In order to prove the feasibility of the proposed colorimetric sensor, the target ion Pb2+ was tested with the simple sensor. As shown in Figure 1A and B, the dispersed AuNPs (12 nm in diameter) showed an obvious UV-vis absorption peak at 520 nm, while the aggregated AuNPs caused a red shift of the absorption peak, and finally formed a new peak at 635 nm. In this work, the absorbance ratio of A520/A635 is chosen to quantify the AuNPs aggregation which causes color change. The high ratios indicate red dispersed AuNPs, while low ratios represent blue aggregated AuNPs. In the absence of Pb2+, STM was not cleaved; the well-assembled HCR products could not protect the AuNPs against salt-induced aggregation (Figure 1C). Therefore, AuNPs were aggregated and a blue color was shown. Conversely, in the presence of Pb2+, split STM can’t trigger HCR effectively, so AuNPs were stabilized

by DNA hairpins (Figure 1D) and maintained the red color.

Figure 1 (A) UV-vis absorption spectra of the colorimetric sensor. Inset showed the visualization results of colorimetric sensor. (B) TEM image of the unmodified AuNPs, diameter was calculated based on the statistical results of 1000 AuNPs. (C) TEM image of the aggregated AuNPs (0 nM Pb2+ was tested). (D) TEM image of the dispersed AuNPs (15 nM Pb2+ was tested). Comparison of colorimetric sensors with or without the stage of HCR signal amplification. It is believed that HCR can make the color change more remarkable and significantly improve the sensitivity of the proposed colorimetric sensor. To verify the significant performance improvements, a simple colorimetric sensor without HCR signal amplification was constructed for comparison (both were carried out under the optimum conditions). As shown in Figure 2A, it is clear that DNA conformation changes significantly with the help of HCR even at the low Pb2+ concentrations (with the increase of Pb2+ concentration, the DNA band representing HCR products is obviously converted to that representing DNA hairpins). This is because each trigger DNA (STM) can assemble a large number of DNA hairpins into long dsDNA. By carefully adjusting the concentration of STM, a small amount of STM fragmentation can effectively reduce the assembly of DNA hairpins, resulting in significant changes in DNA conformation. Conversely, as shown in Figure 2B, without the help of HCR, each RNA-cleavage reaction produces only minor changes in DNA conformation (the DNA bands representing the STM is converted to ST and SM only when the Pb2+ concentration is high enough). As a result, as shown in Figure 2C, with the help of HCR, more obvious color change is observed, which is more convenient for quantitative analysis by naked eye. For the limit of detection (LOD) calculated based on the UV-vis absorbance ratio A520/A635, the



colorimetric sensor with HCR also showed obvious advantages (Figure 2D), the LOD of 59.39 pM is 70 times lower than that of 4.19 nM. According to these results, it can be concluded that the HCR signal amplification strategy applied in this study does improve the sensitivity and is conducive to signal readout. Figure 2 (A) PAGE results of the colorimetric sensor with HCR. DNA conformation changes from HCR products to DNA hairpins with the increase of Pb2+ concentration. (B) Denatured PAGE results of the colorimetric sensor without HCR. DNA conformation changes from STM to ST and SM with the increase of Pb2+ concentration. (C) Visualization results of colorimetric sensors with or without HCR. (D) Relationship between Pb2+ concentration and A520/A635 established by colorimetric sensors with or without HCR. Optimization of HCR substrate sequences. As a simple and effective signal amplification strategy, the efficiency of HCR is critical to the performance of the proposed Pb2+ colorimetric sensor. The DNA hairpins used in this study were designed according to the simple HCR design principles proposed by Pierce and coworkers.40 According to the simple design principle, when the trigger DNA (STM) is divided into two functional domains at the ribonucleotide site, most of the DNA hairpin sequences are fixed, and only the loop sequence of HA and the toehold sequence of HB are uncertain (Figure S1A). In order to obtain better performance, the website software NUPACK (www.nupack.org)58, 59 is used to adjust these uncertain sequences and simulate the DNA assembly results (Figure S1B). As a result, two groups of DNA hairpins were designed and compared. In the absence of Pb2+, STM was not cleaved (Figure S2A, lane a), therefore most of the DNA hairpins were well assembled (Figure S2B, lane 1, 3, 5, and 7). Conversely, when STM was split into ST and SM in the presence of Pb2+ (Figure S2A, lane b), DNA hairpins did not assembled and maintained the hairpin structure (Figure S2B, lane 2, 4, 6, and 8). As the most significant difference in DNA assembly was found between the lane 7 and lane 8, the

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combination of HA8 and HB8 was used in this Pb2+ colorimetric sensor for signal amplification. In addition, in order to improve the dissociation of STM to improve the HCR efficiency, a DNA strand (cGR5) completely complementary to GR5 is used. In this work, cGR5 has a similar DNA sequence with the correct trigger DNA (STM). However, the separation of functional domains in cGR5 greatly reduces its ability to initiate HCR, and will not cause an increase of background signal. As shown in Figure S3, on the one hand, the GR5, cGR5 or GR5+cGR5 can’t initiate HCR in the absence of STM. On the other hand, the addition of cGR5 can significantly improve the HCR efficiency (comparison between lane 8 and lane 9 in Figure S3). Accelerating HCR by DNA annealing. In this study, HCR signal amplification provides significant advantages for the colorimetric detection, but it is also a timeconsuming process,41, 44, 60 which will prolong the total time of Pb2+ detection and is not conducive to rapid detection. Although some organic solvents have been reported to accelerate DNA assembly,61 the strategy is not suitable for the colorimetric sensor here because the organic solvents will affect AuNPs aggregation and colorimetric detection. Figure 3 DNA hairpins are assembled through two different ways. Mixed solutions containing 200 nM HA8, 200 nM HB8, and 20 nM STM, which were slowly cooling down with boiling water for 30 min or incubated at room temperature for hours, control was the mixture without STM, which was also slowly cooling down with boiling water. (The amount of remaining DNA hairpins was quantified by the software GelAnalyzer and used to fit curves.) In this study, we found that a simple operation of DNA annealing can significantly accelerate the process of HCR, while maintaining a low background in the absence of trigger DNA (Figure 3). We suspect that this is benefiting from the well-designed metastable structure of DNA hairpins. On the one hand, DNA hairpins are opened at high temperature, and the collision probability

between DNA molecules also increases, resulting in a distinct acceleration of HCR process. On the other hand,


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since the stem sequences are complementary and linked by short loop domain, in the absence of trigger DNA, it is much easier to form the DNA hairpin structure in the same DNA molecule than to form the dsDNA structure between different DNA molecules. Therefore, annealingaccelerated HCR can significantly shorten the reaction time while maintaining a low background signal (detail mechanism is still under study). As a result, through DNAzyme mediated RNA-cleavage and annealingaccelerated HCR, Pb2+ detection can be completed in about 30 min by the proposed colorimetric sensor. Optimization of the STM concentration. After designing and optimizing the DNA hairpin sequences, the STM concentration was further optimized.

Figure 4 (A) Different concentrations of STM were used to initiate HCR in absence (-) or in presence (+) of 500 nM Pb2+ (B) UV-vis absorbance ratio when different concentrations of STM were used. Inset showed the visualization results. (C) Different volume of DNA mixture was added in 100 μL AuNPs, the colorimetric results were adjusted to have similar background for comparison by adding different volume of TrisHAc. Inset showed the visualization results. (D) Different volume of TrisHAc was added after the addition of 7.5 μL of DNA mixture to adjust the absorbance ratio. Inset showed the visualization results. As shown by PAGE results in Figure 4A, DNA hairpins can be well assembled when HCR was initiated by 25 nM or higher STM concentration. Besides, the PAGE results also showed that HCR products tended to shorten when excessive trigger DNA was used to initiate HCR, which was consistent with the reported articles.40, 48, 62 This is because each trigger DNA molecule can initiate HCR equally, while over trigger DNA can result in the lack of DNA hairpins, thereby shortening the HCR products.40 In this simple colorimetric sensor, the unassembled DNA hairpins can stabilize AuNPs against salt-induced aggregation, while the well assembled HCR products can hardly stabilize AuNPs. However, although the unassembled DNA hairpins decreased with the increase of STM concentration (Figure 4A), the AuNPs

did not well aggregate when HCR was initiated by excessive (75 nM or 100 nM) STM (Figure 4B). This is because each HCR product has short unpaired ssDNA tails, which can also stabilize AuNPs to some extent. Therefore, as excessive STM made the HCR products shorter in length but larger in quantity, the absorbance ratio (A520/A635) of background (0 nM Pb2+ was tested) tends to be higher. On the other hand, the absorbance ratio of signal (500 nM Pb2+ was detected) was weakened when excessive STM was used (Figure 4B). This is because more unsplit STM is left when excessive STM is used, which can initiate the HCR and reduce the absorbance ratio of signal. As a result, the UV-vis absorbance ratio difference (ΔA520/A635) between signal and background decreased when excessive STM was used, which was detrimental for improving the sensitivity of the colorimetric sensor. According to the results shown in Figure 4A and B, the STM concentration of 25 nM was used to construct the Pb2+ colorimetric sensor in this work. Optimization added DNA mixture and TrisHAc volume in the stage of visualization signal generation. Since more DNA can stabilize AuNPs against saltinduced aggregation to a greater extent, the volume of DNA mixture use for colorimetric detection will affect the colorimetric performance. As shown in Figure 4C, when 500 nM Pb2+ was tested, the difference of absorbance ratio (ΔA520/A635) reached the maximum when 7.5 μl of DNA reaction mixture was added into 100 μl of AuNPs for colorimetric detection. In this work, DNAzyme mediated RNA-cleavage and HCR signal amplification were performed in the reaction buffer with certain ionic strength (final concentration of 100 mM NaAc, 10 mM TrisHAc (pH 7.5) and 1 mM EDTA). However, it was found that this ionic strength was too weak to achieve good colorimetric performance. Because the color change caused by AuNPs aggregation is more obvious in a certain range, the addition of TrisHAc (100 mM, pH 7.5) needs to be carefully adjusted. As shown in Figure 4D, the absorbance ratio of both signal and background decreased with the increase of the TrisHAc addition. Moreover, the difference of absorbance ratio (ΔA520/A635) reached the maximum when 5 μL of TrisHAc was added. However, as shown by the visualization results (Figure 4D inset), more significant color change was observed when 5.5 μL or 6 μL TrisHAc was added. In order to facilitate the visualization detection, 5.5 μL of TrisHAc was added to adjust the colorimetric detection all through this work. Reaction time of the RNA-cleavage reaction. In order to save the whole time of Pb2+ detection, the simple operation of annealing was used to shorten the reaction time of HCR. In addition, the incubation time of RNA-cleavage was also DNAzyme mediated investigated. Benefiting from the significant signal enhancement caused by HCR, small changes in the amount of STM can trigger large changes in DNA conformation and result in obvious color change. Therefore, only a small amount of STM needs to be



cleaved, and the DNAzyme mediated RNA-cleavage can be completed in about 10 min (Figure S4). Sensitivity of the proposed Pb2+ colorimetric sensor. After optimizing serious reaction conditions, different concentrations of Pb2+ were tested under the optimum condition (as shown in the experimental section).

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found that the absorbance ratio showed good linear relationship with Pb2+ concentration at both lower (0~1 nM) or higher Pb2+ concentration (1~15 nM), the linear equations are shown in the Figure 5B. In this work, the segmented standard curve may be due to the fact that the aggregation of AuNPs is not completely linearly correlated with the ratio of A520/A635. Just as the aggregation of AuNPs has more significant color change in a certain range, and the slope of linear correlation may be different in different ranges. However, it is believed that accurate detection results can be easily obtained by using the corresponding standard curve in different ranges. And the limit of detection of 59.39 pM was achieved (LOD, defined as 3σ/S, σ refers to the standard deviation of the blank samples, and S refers to the slope of the fitting standard curve). As can be seen from Table S2 ， the proposed colorimetric sensor has achieved well performance in comparing with some other reported sensors. Selectivity of the proposed Pb2+ colorimetric sensor. The selectivity of the proposed colorimetric sensor is mainly attributed to the application of highly specific DNAzyme. It is reported that the DNAzyme GR5 has remarkable specificity with Pb2+.32 In this study, some other metal ions including Ca2+, Mn2+, Cd2+, Ba2+, Zn2+, Fe3+, Ni2+, Cu2+, Cr3+, and Hg2+ were tested to evaluate the selectivity of the constructed colorimetric sensor. As shown in Figure 6, other metal ions can hardly generate response of the colorimetric sensor even at a much higher concentration (500 times the concentration of Pb2+). Moreover, the absorbance ratio was almost unchanged in the presence of the mixture of other ions, thus other metal ions have little influence on Pb2+ detection. According to these experimental results, the proposed Pb2+ colorimetric sensor shows good selectivity and can resist the interference from other metal ions.

Figure 5 (A) UV-vis absorption spectra of the colorimetric detection results under the optimum conditions. (B) Relationship between detected Pb2+ concentration and A520/A635 established by the proposed colorimetric sensor with HCR signal amplification (linear relationship is shown in the dotted box). As shown by the PAGE results in Figure 2A, HCR was weakened and more DNA hairpins were not assembled as the concentration of Pb2+ increased. Due to the unassembled DNA hairpins can stabilize AuNPs, the AuNPs aggregation degree decreased with the increase of Pb2+ concentration, and the visualization results showed that the color of AuNPs changed from blue to red gradually (Figure 2C). In order to show the AuNPs aggregation more accurately, the UV-vis spectra were recorded (Figure 5A), and the absorbance ratio (A520/A635) was calculated to quantify the AuNPs aggregation that causing color variation. As a result, as shown in Figure 5B, the absorbance ratio increased with the increase of Pb2+ concentration. In addition, it was also

Figure 6 Selectivity of the proposed colorimetric sensor towards Pb2+. Inset showed the visualization results of detecting other metal ions or target ion. (10 μM of the other metal ions, 20 nM of Pb2+, and their mixture were tested. All data were of five independent repeats.)


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Actual sample detection. In addition to high sensitivity and good selectivity, practical applicability of the proposed Pb2+ colorimetric sensor was also investigated. Because only a small volume of DNA mixture is used for colorimetric detection, it is anticipated that the impurities in actual water samples have little influence on AuNPs aggregation and colorimetric detection. Table 1. Analysis of Pb2+ in actual water samplesa (n=5). Sample

Original (nM)b

ICP-AES

Added (nM)

Found (nM)

ICP-AES

Recovery (%)

Tap water

0.298*2±0.022

N.D

2.5

2.950±0.129

2.923±0.140

106.09

Surface rain

0.134*2±0.011

N.D

5.0

5.130±0.199

5.32±0.318

99.91

Pond water

0.883*2±0.084

1.700±0.117

10.0

10.736±0.454

10.925±0.505

98.54

[a] The mean concentrations were calculated by the linear curve of the proposed Pb2+ colorimetric sensor. [b] Dilution factor was 2, because 50 μL of actual water samples were tested in a total reaction volume of 100 μL.

In the actual sample detection, three actual water samples were centrifuged and filtered to remove the insoluble impurities at first, and then the original Pb2+ concentration of the actual samples were tested by the colorimetric sensor. In order to calculate the recovery of the proposed colorimetric sensor, different concentrations of Pb2+ were tested in the presence of actual water samples, and the recoveries of 106.09%, 99.91% and 98.54% were achieved respectively (Table 1). According to these results, it can be concluded that this simple colorimetric sensor can be applied to the detection of actual water samples.

The DNA oligonucleotides, reagents and apparatus used in this study, some experimental operations, additional figures and comparison of the proposed method with other methods (PDF)

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

ORCID Yixiang Duan: 0000-0001-8346-0421

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

CONCLUSIONS In summary, an enzyme-free, label-free and highly sensitive colorimetric sensor was proposed for Pb2+ detection. The annealing accelerated HCR provided a more obvious color change and higher sensitivity by causing more significant DNA conformational changes, which can be used to detect targets due to the widely application of DNA mediated RNA cleavage reaction22. Moreover, by using unmodified AuNPs, label-free DNA, and HCR, the proposed colorimetric sensor avoids any modification or complex enzymatic reactions, thus facilitating the operation and reducing the costs. Considering the high specificity and practical application are both achieved, the colorimetric sensor is suitable for environmental monitoring and food safety assessment.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was gratefully supported by the National Natural Science Foundation of China (No. 21874095).

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