Highly sensitive and selective colorimetric sensors for uranyl (UO2(2+)): development and comparison of labeled and label-free DNAzyme-gold nanoparticle systems

Abstract

Colorimetric uranium sensors based on uranyl (UO2(2+)) specific DNAzyme and gold nanoparticles (AuNP) have been developed and demonstrated using both labeled and label-free methods. In the labeled method, a uranyl-specific DNAzyme was attached to AuNP, forming purple aggregates. The presence of uranyl induced disassembly of the DNAzyme functionalized AuNP aggregates, resulting in red individual AuNPs. Once assembled, such a "turn-on" sensor is highly stable, works in a single step at room temperature, and has a detection limit of 50 nM after 30 min of reaction time. The label-free method, on the other hand, utilizes the different adsorption properties of single-stranded and double-stranded DNA on AuNPs, which affects the stability of AuNPs in the presence of NaCl. The presence of uranyl resulted in cleavage of substrate by DNAzyme, releasing a single stranded DNA that can be adsorbed on AuNPs and protect them from aggregation. Taking advantage of this phenomenon, a "turn-off" sensor was developed, which is easy to control through reaction quenching and has 1 nM detection limit after 6 min of reaction at room temperature. Both sensors have excellent selectivity over other metal ions and have detection limits below the maximum contamination level of 130 nM for UO2(2+) in drinking water defined by the U.S. Environmental Protection Agency (EPA). This study represents the first direct systematic comparison of these two types of sensor methods using the same DNAzyme and AuNPs, making it possible to reveal advantages, disadvantages, versatility, limitations, and potential applications of each method. The results obtained not only allow practical sensing application for uranyl but also serve as a guide for choosing different methods for designing colorimetric sensors for other targets.

Figure 1

(A) The scheme of labeled colorimetric sensor based on AuNP disassembly in the absence and presence of UO₂²⁺. In the presence of UO₂²⁺, the length of the weakest complementary part in the aggregates becomes shorter due to UO₂²⁺ induced substrate cleavage. The substrate cleavage can decrease melting temperature of AuNP aggregates. A_n in the arm strands indicates 0A or 12A spacers (n=0 or 12). (B) As UO₂²⁺ is introduced into AuNP aggregates and temperature is controlled above the melting temperature of UO₂²⁺ treated aggregates, AuNP disassembles. (C) ³²P assay result showing the cleavage kinetics in the presence of UO₂²⁺. (D) The melting curve of A₁₂ aggregates with (blue curve) and without (red curve) UO₂²⁺. There is about 10 ° C decrease of melting temperature in the presence of UO₂²⁺. bps=base pairs.

Figure 2

The schematic design of the labeled sensor including the sequences of the invasive DNAs (pink) and 0A, 12A, and 24A Arm strands (blue and red) used in this work.

Figure 3

The background increase of A₀, A₁₂, and A₂₄ aggregates with invasive DNA strands in the absence of UO₂²⁺. (B) Disassembly kinetics difference between A₀, A₁₂, and A₂₄ aggregates in the presence of Inva-6. The effect of Inva-6 on the UO₂²⁺ induced disassembly of A0 (C) and A₁₂ (D) aggregates. The effect of longer invasive DNAs on A₁₂ (E) and A₂₄ (F) aggregates. UO₂²⁺ was added to samples one minute after UV-vis monitoring was started.

Figure 4

(A) Disassembly of AuNP aggregates at various UO₂²⁺ concentrations and (B) calibration curve of labeled uranyl colorimetric sensor. (C) Disassembly of AuNPs in the presence of various metal ions including UO₂²⁺. (D) The color change of AuNP aggregates in the presence different concentrations of UO₂²⁺ and other metal ions. The concentration of all metal ions other than UO₂²⁺ is 2 μM. Metal ions were added to samples one minute after UV-vis monitoring was started.

Figure 5

(A) The design and sequence of the label free sensor (complex). After UO₂²⁺ induced cleavage, 10 mer ssDNA is released which adsorbs on AuNP surface. (B) AuNP reaction in addition of UO₂²⁺ treated/untreated complex and additional NaCl. AuNPs aggregate in the absence of UO₂²⁺ but remain dispersed in its presence.

Figure 6

(A) Extinction ratio dependence on the number of 10 mer ssDNA per 13 nm AuNP. The stability of AuNP increases as more ssDNA exist per one AuNP. (B) The color change of AuNPs with different ratio of DNA per AuNP.

Figure 7

(A) The quenching efficiency of label free sensor by shifting the pH of the solution from 5.5 to 8. AuNPs are aggregated in the absence of UO₂²⁺ (blue curve) but remains dispersed after 6 minutes reaction with 2 μM UO₂²⁺ (red curve). AuNPs show less amount of disassembly after 1 minute of reaction with 2 μM UO₂²⁺ and quenching (purple curve). No further disassembly of AuNP aggregates after 5 minutes of holding in between quenching and AuNP addition (green curve) shows that quenching efficiency is very high and quick. The color change of each sample is also shown in the inset. (B) The color change difference before (upper) and after (lower) addition of TRIS base solution. Concentration of uranyl is 2 μM.

Figure 8

(A) Calibration curve of label free sensor. Sensor has detection limit of 1 nM. (B) The color change of AuNP solution with different concentration of UO₂²⁺ in the solution. (C) The color change of AuNP solution with various metal ions including UO₂²⁺ (2 μM).