Precisely Determining Ultralow level UO22+ in Natural Water with Plasmonic Nanowire Interstice Sensor

Uranium is an essential raw material in nuclear energy generation; however, its use raises concerns about the possibility of severe damage to human health and the natural environment. In this work, we report an ultrasensitive uranyl ion (UO22+) detection method in natural water that uses a plasmonic nanowire interstice (PNI) sensor combined with a DNAzyme-cleaved reaction. UO22+ induces the cleavage of DNAzymes into enzyme strands and released strands, which include Raman-active molecules. A PNI sensor can capture the released strands, providing strong surface-enhanced Raman scattering signal. The combination of a PNI sensor and a DNAzyme-cleaved reaction significantly improves the UO22+ detection performance, resulting in a detection limit of 1 pM and high selectivity. More importantly, the PNI sensor operates perfectly, even in UO22+-contaminated natural water samples. This suggests the potential usefulness of a PNI sensor in practical UO22+-sensing applications. We anticipate that diverse toxic metal ions can be detected by applying various ion-specific DNA-based ligands to PNI sensors.

Uranium, a representative radioactive metal, has been used as the main source of nuclear power generation via nuclear fission 1 . Uranium can exist in various forms in the environment, such as uranyl fluoride (UO 2 F 2 ), uranyl tetrafluoride (UF 4 ), uranium dioxide (UO 2 ), and triuranium octoxide (U 3 O 8 ). The accumulation of these compounds in the human body can lead to severe health problems 2 . For example, UO 2 F 2 and UF 4 cause kidney damage, and UO 2 and U 3 O 8 can cause certain cancers and mutations by accumulating in the lungs 3 . Uranium is also found in diverse forms in aqueous conditions. The uranyl ion (UO 2 2+ ) is the most soluble and common form 4,5 . Because UO 2 2+ can disturb organ function by accumulating in the skeleton, kidneys, lungs, and liver 6,7 , the detection of UO 2 2+ in natural water is very important. Traditionally, UO 2 2+ has been detected using various physical and chemical techniques, including inductively coupled plasma-mass spectrometry 8 , radio spectrometry 9 , atomic adsorption spectrometry 10 , and phosphorimetry 11 . However, these methods present several drawbacks in that they require expensive instruments, involve complicated methods, and are time-consuming. Recently, nanomaterial-based UO 2 2+ sensing methods have been developed by employing fluorescence 12,13 , electrochemistry 14 , resonance scattering 15,16 , colorimetry 17 , magnetoelasticity 18 , and surface-enhanced Raman scattering (SERS) [19][20][21] . Among these techniques, SERS offers highly sensitive molecular detection because SERS signals can be obtained from a small number of molecules or even a single molecule located within a sub-10 nm metallic nano-gap (hot spot)  . This technique also provides molecular fingerprints and causes less photobleaching [43][44][45][46][47][48] . Nevertheless, there are only a few reported SERS sensors for UO 2 2+ detection, and most of them have not been validated in real environments. Therefore, further improvements in sensitivity, selectivity, and reproducibility are still needed for the practical sensing of UO 2 2+ in a real environment using a SERS sensor.
Here, we report an ultrasensitive UO 2 2+ detection method using a plasmonic nanowire (NW) interstice (PNI) sensor combined with a DNAzyme-cleaved reaction. SERS-based sensing methods are often affected by uncontrolled aggregation and size distribution of nanomaterials, which are detrimental to the reproducibility of the sensor [49][50][51] . A PNI platform composed of a single-crystalline Au NW with an atomically flat surface has shown improved sensitivity and reproducibility 31 . By combining the DNAzyme cleaving method with a PNI platform, we quantitatively detected UO 2 2+ with an ultralow detection limit of 1 pM and high selectivity. More importantly, the precise detection of UO 2 2+ was demonstrated in several UO 2

2+
-contaminated natural water samples, showing the practical applicability of the PNI sensor.

Results and Discussion
DNA-based ligands, including aptamers and DNAzymes, have been widely used to detect various metal ions because they provide several advantages, such as high affinity, selectivity, stability, and relative ease of modification 52,53 . In this experiment, we used a DNAzyme, which has the sequence shown in Fig. 1a, for the detection of UO 2 2+ . A DNAzyme is composed of an enzyme strand and a substrate strand. The enzyme strand (green) is a specific sequence that can bind to UO 2

2+
. The substrate strand (blue) is hybridized with the enzyme strand. In the middle of the substrate strand, a ribose-adenosine (rA) sequence (red) is present. Ribonucleotides are approximately 100,000-fold more susceptible to hydrolytic cleavage than deoxyribonucleotides 13 . At the 5′ end of the substrate strand, Cy5 (magenta) is present. Cy5 is a well-known Raman reporter for the 633 nm excitation source [27][28][29][30][31][32] . When UO 2 2+ was added to DNAzyme, UO 2 2+ bound to the enzyme strand, and thus, the rA sequence on the substrate strand was hydrolytically cleaved. The cleaved strand was released from the DNAzyme and then captured by a PNI sensor. The PNI sensor was prepared by laying down a single-crystalline Au NW onto a Au film 24 (Fig. 1b). Au NWs were synthesized using a previously reported vapor transport method 54 and then modified by the addition of a complementary capture DNA strand to the cleaved strand. The length of capture strand was set as 15 base pairs to increase the hybridization efficiency with cleaved strand 55 . The PNI sensor with an attached cleaved strand can provide a strong Cy5 SERS signal, enabling the detection of UO 2 2+ . The left panel in Fig. 1b shows the result of using a PNI sensor combined with a DNAzyme-cleaved reaction for UO 2 2+ detection. When UO 2 2+ was present in the sample solution at the concentration of 10 nM, 4 major bands at 1185, 1360, 1485, and 1580 cm −1 were clearly detected by the PNI sensor (red spectrum). These bands correspond to the v(C-N) stretch , v(C = C) ring , v(C-C) ring , and v(C = N) stretch of Cy5, respectively 28 . In contrast, when UO 2 2+ was absent from the sample solution, the PNI sensor exhibited a weak SERS signal (blue spectrum). In this way, UO 2 2+ can be detected from the turn-on of the SERS signal. Generally, turn-on type sensors are more suitable than turn-off type sensors for practical applications because they result in fewer false-positive results 17 . Figure 2a shows the SERS signals measured from the PNI sensors while varying the UO 2 2+ concentration from 1 pM to 100 nM. The samples were prepared by a 1/10 serial dilution, and a blank sample solution was prepared as a control. In the blank sample solution, the PNI sensor provided a weak Cy5 SERS signal (black spectrum in Fig. 2a). In the 1 pM sample, the signal was significantly enhanced (red spectrum in Fig. 2a). Figure 2b shows the intensity of the Cy5 1580 cm −1 band plotted as a function of the UO 2 2+ concentration. The SERS signal from the PNI sensor increased as the UO 2 2+ concentration increased, saturating at a concentration of 10 nM. The inset in Fig. 2b shows the dynamic range of the PNI sensor for UO 2 2+ detection. The SERS signal intensity linearly increased within the UO 2 2+ concentration range of 1 pM to 10 nM. This wide dynamic range makes this technique advantageous for the quantitative detection of UO 2 2+ . We estimated the detection limit of the PNI sensor to be 1 pM. This detection limit is approximately 1,000-fold lower than the limits of other SERS-based sensors 21 . Furthermore, the detection limit of this method is comparable to that of the most sensitive detection method, which is based on a resonance scattering spectral technique 15 .
To confirm the selectivity of a PNI sensor combined with a DNAzyme-cleaved reaction, SERS signals were observed from the PNI sensors after mixing DNAzyme with samples containing various metal ions (UO 2 2+ , Cd 2+ , Hg 2+ , Pb 2+ , Ca 2+ , Mg 2+ , Zn 2+ , Cu 2+ , Fe 2+ , Co 2+ , Ni 2+ , and Th 4+ ). The concentration of each metal ion was 10 nM. Figure 3 shows the intensity of the Cy5 1580 cm −1 band measured from the PNI sensors for detecting the various (b) Schematic illustration of UO 2 2+ detection by a PNI sensor combined with a DNAzyme-cleaved reaction. The left panel shows the SERS spectra measured from PNI sensors in the absence of (blue spectrum) and the presence of 10 nM UO 2 2+ (red spectrum).
metal ions. Remarkable SERS signal was observed only in the presence of UO 2 2+ (magenta bar in Fig. 3), and no distinct SERS signal was observed in the presence of other metal ions (cyan bars in Fig. 3). These results indicate that the proposed detection method is highly specific for UO 2 2+ .  Finally, we examined the applicability of a PNI sensor combined with a DNAzyme-cleaved reaction to detect UO 2 2+ in natural water. The natural water samples were obtained from various environments, including a sea, a river, a lake, and a tap. Buffer solution was also prepared as a control. Figure 4 shows the intensities of the Cy5 1580 cm −1 band measured from the PNI sensors after mixing DNAzyme with the natural water samples. The magenta bars represent the natural water samples spiked with UO 2 2+ and the cyan bars represent the as-collected natural water samples. Strong SERS signals were observed only after the addition of UO 2 2+ into the natural water samples. Without UO 2 2+ , SERS signals were rarely observed. Note that the DNAzyme is sensitive to ionic strength 56 . The DNAzyme is in the lock-and key mode at the ionic strength of 100 mM or higher 56 . In addition, the concentration of Mg 2+ can affect the DNAzyme activity 56 . At the lower concentration of 2 mM, the activity is promoted. On the other hand, the activity is inhibited at the higher concentration of 2 mM. In this experiment, the DNAzyme solution and natural water sample were mixed with 9:1 volume ratio before the SERS measurement. The ionic strengths of the mixtures are calculated as 246.43 mM (sea), 131.81 mM (river), 134.72 mM (lake), and 132.07 mM (tap) according to the inductively coupled plasma-optical emission spectroscopy (ICP-OES) and ion chromatography (IC) data of natural water samples (Table S1 and S2 in supplementary information). The concentrations of Mg 2+ in the mixtures are 1.31 mM (sea), 0.01 mM (river), 0.02 mM (lake), and 0.01 mM (tap). Since the ionic strengths of mixtures are higher than 100 mM and the concentrations of Mg 2+ in the mixtures are below 2 mM, the PNI sensor combined with a DNAzyme-cleaved reaction can detect UO 2 2+ successfully even in natural water samples. The USA's limit for UO 2 2+ in drinking water is 30 μ g/L (approximately 100 nM), however, the long-term consumption and exposure to water that contains UO 2 2+ , even at concentrations below this limit, can cause severe toxicity and serious diseases 57 . We anticipate that the present method can be employed for practical UO 2 2+ detection and aid in the prevention of environmental pollution and human diseases caused by UO 2 2+ .

Conclusion
We detected UO 2 2+ by combining an ultrasensitive PNI platform with a DNAzyme-cleaved reaction. The DNAzyme specifically reacted with UO 2 2+ and released a cleaved strand. The PNI sensor sensitively captured the cleaved strand, which enabled the detection of UO 2 2+ . We quantitatively detected UO 2 2+ with an ultralow detection limit of 1 pM and high selectivity. Moreover, this method enables the detection of UO 2 2+ in various natural water sources, such as sea, lake, river, and tap. A PNI sensor that can precisely detect small quantities of UO 2 2+ in natural water is expected to reveal many environmental pollutants, and hence minimize damage to the human body caused by UO 2 2+ exposure.

Synthesis of single-crystalline Au NWs.
Single-crystalline Au NWs were synthesized on a c-cut sapphire substrate in a horizontal quartz tube furnace system following the chemical vapor transport method described in a previous report 54 . An alumina boat containing an Au powder was positioned directly below the heat source. The sapphire substrate was placed a few centimeters downstream from the alumina boat. The heating zone was brought to 1100 °C while the chamber pressure was maintained at 3-5 Torr. Ar gas flowing at 100 sccm was used to transport the Au vapor. Au NWs were grown on the sapphire substrate over a 1 h period.
Preparation of PNI sensors. Au film substrates were prepared on Si substrates by electron beam-assisted deposition of a 10 nm thick film of Cr followed by a 300 nm thick film of Au. The prepared Au films were SERS-inactive by themselves 24 . The Au films were then cut to 1 cm 2 for PNI sensor fabrication. To prepare the capture probe DNA-attached Au NWs, as-synthesized NWs were incubated with 5 mM captured DNA in 1 M KH 2 PO 4 buffer (pH 6.75) at room temperature for 12 h. Next, the Au NWs were rinsed with a 0.2% (w/v) SDS solution for 5 min. The capture probe DNA-attached Au NWs were then transferred onto Au films by a simple attachment and detachment process 30 . Briefly, the NW-grown c-cut sapphire substrates were inverted onto Au film substrates containing medium. Before both substrates were overlapped, a drop of distilled water was applied as a lubricant. The sapphire substrates were then pushed gently and, after a few seconds, detached. After the attachment and detachment process, the remaining water was dried under flowing N 2 gas. To stop the enzymatic reaction by shifting the pH of the mixture, 0.1 mM Tris-acetate solution was added. Then, the mixture was dropped onto the PNI sensor and allowed to stand for 2 h. To remove excess DNA, the sensor was rinsed with a 0.2% (w/v) SDS solution for 5 min and then rinsed twice with distilled deionized water.

Detection of UO
Instrumentation. SERS spectra were measured using a micro-Raman system on an Olympus BX41 microscope. A 633 nm He/Ne laser (Melles Griot) was used as an excitation source, and the laser was focused on samples through a 100× objective (NA = 0.7, Mitutoyo). The laser power directed at the sample was 0.4 mW. The SERS signals were recorded with a thermodynamically cooled electron-multiplying charge-coupled device (Andor) mounted on the spectrometer with a 1200 groove/mm grating. The acquisition time for all SERS spectra was 60 s. A holographic notch filter was used to reject laser light. The concentration of metal ion in natural water samples was analyzed by inductively coupled plasma-optical emission spectroscopy (ICP-OES 720, Agilent) and ion chromatography (881 Compact IC pro, Metrohm Ltd.).