Junjie Cheng‡
ab,
Yan Sun‡b,
Lu Zhoub,
Kunchi Zhangb,
Jine Wangb,
Zhengyan Wu*a and
Renjun Pei*b
aKey Laboratory of Ion Beam Bioengineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, People's Republic of China. E-mail: zywu@ipp.ac.cn
bKey Laboratory of Nano-Bio Interface, Division of Nanobiomedicine, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, People's Republic of China. E-mail: rjpei2011@sinano.ac.cn
First published on 27th October 2014
A novel naked-eye distinguishable nanosensor based on phosphorylation triggered poly-nanoparticle assembly strategy has been designed for detecting T4 PNK activity. This colorimetric sensor exhibits conveniently homogeneous operation with simple, sensitive and easily scalable properties.
Gold is the first metal to be transformed into a colloidal state. Its simple synthetic procedure, high extinction coefficients and large surface area make it highly conspicuous in the area of DNA sensing recently.21 Inspired by these advantages, different methods of PNK detection by using gold nanoparticles also have been proposed in recent years. However, the nanoparticles were usually used as signal enhancers but detectors in those methods. For examples, Wang utilized the 5′-phosphate DNA modified gold nanoparticles and the TiO2 nanotubes in order to generate a novel electrochemical sensor for the detection of T4 PNK with Au nanoparticles amplification.15 Huang presented a fluorescence polarization nanosensor based on λ exonuclease cleavage reaction and fluorescence polarization enhancement effect of gold nanoparticles.22 Although these methods were quite effective, they still depended on professional signal detection equipments. After Mirkin and co-workers reported the breakthrough method for detecting polynucleotides which utilizes the distance-dependent optical properties of aggregated gold nanoparticles functionalized with oligonucleotides,23,24 the development of DNA-functionalized gold nanoparticles has been advanced by leaps and bounds. Previous work has demonstrated that large nanoparticles network formed through DNA strands hybridization or ligation can be distinguished by the naked eye.23 In addition, due to the obvious advantages such as larger touch surface, more even reaction and requiring no complex rinse and separation, it seems that homogeneous reaction has an advantage over solid phase reaction. Therefore, it has a wide foreground to introduce the homogeneous reaction in PNK detection system. Here, we have utilized the property of DNA–gold nanoparticles self-assembly to create a simple building block and exploited a homogeneous, highly specific, sensitive, convenient and naked-eye directly identified T4 PNK detection system which has not been reported thus far.
The detection method is illustrated in Scheme 1. The phosphorylation triggered multi-nanoparticle assembly strategy has been designed for detecting T4 PNK activity. There are five sequences (Table 1), two of them (S3 and S4) were immobilized on the surface of gold nanoparticles by strong sulfur–gold adsorption. S1 acting as a target recognition probe and S2 as a common probe, are both designed to be perfectly complementary to S4 and S3 respectively. In the present of T4 PNK, the 5′-hydroxyl termini of S1 can be phosphorylated, and the subsequent ligation process mediated by T4 DNA ligase will process and produce a complete long strand S12. Hybridization of the newly produced oligonucleotide S12 with the two probes on gold nanoparticles results in a gradual color fade and a precipitation reaction ensues within 30 minutes. In order to improve the efficiency and specificity of ligation, hS1S2 is introduced as a helper strand which contains a 5-base-pair overlap sequence with S1 and another 5-base-pair overlap with S2. The 10-base oligonucleotide closes S1 and S2 through hybridization and causes the relatively high efficiency of the two adjacent probes covalently joining by T4 DNA ligase. Since the probe S2 also carries the 5′-hydroxyl termini, it will be added into the reaction solution after S1 was phosphorylated by T4 PNK to avoid the undesired target-catalyzed phosphorylation. In the assembly step, S3 and S4 have much more complementary bases with S12 than hS1S2 so that ssDNA S12 prefers to hybridize with the probes on the nanoparticles and forms a more stable double strand, rather than with the helper hS1S2.25 Nevertheless, in the absence of T4 PNK, S1 keeps its 5′-hydroxyl termini so that T4 DNA ligase cannot perform its function. Although S1 and S2 can still hybridize with S3 and S4, but never link together. Accordingly, it can hardly form the poly-DNA–gold nanoparticle assembly.
![]() | ||
Scheme 1 Sketch to the concept and the experimental principle of PNK detection using gold nanoparticles. |
DNA code | DNA sequence |
---|---|
S1 | 5′-GCA GGA CCA TTC TTT GAT ACA GA-3′ |
S2 | 5′-ACT AGC TAC GAT-3′ |
CK | 5′-ACT AGC TAC GAT GCA GGA CCA TTC TTT GAT ACA GA-3′ |
S3 | 5′-C6S-S-TCT GTA TCA AAG AAT GGT CCT GC-3′ |
S4 | 5′-ATC GTA GCT AGT-C3S-S-3′ |
hS1S2 | 5′-CCT GCA TCG T-3′ |
We first validated the feasibility and effectiveness of the T4 PNK detection system by directly colorimetric response in Fig. 1A. The left sample of the photograph shows distinctly polymeric gold nanoparticle–polynucleotide aggregate and highly transparent, colorless supernatant in standardized detection system. It indicates the successful phosphorylation of the target recognition probe by T4 PNK. In contrast, no apparent precipitation is observed in the middle and right ones even after prolonged storage, which represent adding inactive PNK and without PNK respectively. It has proved that this analytical assay is as simple and effective as we initially predicted. Furthermore, it is worthwhile to note that most of the previous works showed a red to blue or gray color change in their experimental solutions during detection process, which are different from our purplish red aggregates.24,26–31 Since nanoparticle aggregates with interparticle distances substantially greater than the average particle diameter appear red, but as the interparticle distances in these aggregates decrease to less than approximately the average particle diameter, the color becomes blue.24,32,33 We can imply that the interparticle distance is still slightly greater than or close to the average particle diameter here.
As is well known, in most systems, the gold nanoparticles in solution were stabilized by the adsorbed negative ions (citrate). Their repulsion prevented the strong van der Waals attraction between gold nanoparticles from causing them to aggregate. However, high concentration of salts will screen the charge on the surface of gold nanoparticles, resulting in the gold surface plasmon state change, and color becomes blue and nanoparticles aggregate.34,35 This would be difficult to distinguish whether the color change is caused by the target or high ionic strength. Fortunately, the result of our detection system is credible, regardless of considering the problems above, because the DNA hybridization just leads to the precipitate but color change.
Moreover, the phosphorylation process is further confirmed by analyzing the reaction products in an agarose gel electrophoresis (AGE), Fig. 1B. The slowly and rapidly migrating nucleotide fragments have appeared in lane 2 and lane 3, which displayed the ligation results of weather phosphorylated or not respectively. The results clearly indicate that T4 PNK acts as a necessary prerequisite for ligation during repair, inducing T4 DNA ligase repairing DNA nicks and forming a complete longer probe. The insensitivity of AGE allows the palpable observation of DNA bands that are longer than about 20 bases. There is a clear single band of the ligated S12 with helper hS1S2 in lane 2. Consequently, for the failure ligation sample, nothing but the band of S1 (23 bases) will be seen in lane 3 (S2 only has 12 bases). In addition, the band in lane 2 is much brighter than the one in lane 3. We suspected the explication that SYBR Green I presents much higher affinity to double strands than single strand DNA. As joining of nicks by DNA ligase, the hybridization of helper sequence hS1S2 with S12 is more stable and the duplex contains 10 complementary base pairs. Otherwise hS1S2 may be released from the two shorter strands (S1 and S2) during the electrophoresis process, and only the single strand S1 can be seen in gel.
To verify the accurate wavelength for UV-vis measurement as well as the positive control purpose, a single strand DNA (CK), whose sequence is same with the ligation product S12 was designed. As shown in Fig. 2A, the absorbance at 524 nm dramatically decreased with about 20 nm red shift after the addition of CK to the gold nanoparticles collosol directly, which implys the successful hybridization between CK and S3, S4, creating poly-gold nanoparticle assembly.
After having determined the measuring wavelength (524 nm), the kinetics of this assay was followingly explored. The absorbance of each sample was monitored and the time courses were plotted in Fig. 2B. Expect for time gradients, further control experiments were also conducted. The positive control was investigated with CK instead of the ligated S12. Pure water or an inactivated T4 PNK (heated in 75 °C for 10 min) replaced the standardized T4 PNK as two negative controls. As illustrated above, different phosphorylation times showed different kinetic behavior to gold nanoparticle assembly. The absorbance of two negative controls remained approximately constant during whole detection period. Interesting, fairly fast absorbance decreases were revealed for the positive control and most of the phosphorylated products expect for the half hour one, which implys that the successful “phosphorylation and ligation” process of S1 and S2 in the presence of T4 PNK is performed. However, half hour reaction time may be too hasty to phosphorylate enough substrates for linking the gold nanoparticles and 8 hours one seems too long in this system, yet one to two hours are sufficient.
Fig. 3A displayed the UV-vis spectra of the sensing system following the successive addition of T4 PNK. As expected, a gradual decrease in absorbance signal was clearly observed along with the increase of T4 PNK concentration ranging from 0.0385 to 1.3533 U mL−1. It produced a good calibration curve relationship and, more important, the novel method was conducted in PCR tubes which facilitated the operation and was easily available.
It is generally known that a number of factors are likely to influence the activity of the T4 PNK reaction.15,36 Since T4 PNK catalyzes the transfer of the γ-phosphate from ATP to the 5′-OH group of DNA, it is necessary to investigate the influence of ATP concentration on the rate of reaction here. As shown in Fig. 3B, the increase of ATP concentration resulted in the reducing of absorbance responses in low concentration. While a gradually rise was observed when the concentration of ATP continued to increase. This slight inhibition effect is probably due to the competitive binding to T4 PNK between DNA and ATP. When the concentration of ATP is relatively high, the binding sites of T4 PNK for 5′-hydroxyl DNA are partially blocked. Kinetic analysis favors a sequential mechanism in which both ATP and the 5′-OH polynucleotide bind the enzyme prior to dissociation of either product.15,37 Consequently, 0.69 mM was considered as the optimal ATP concentration to obtain a high sensitivity in this detection.
The hS1S2 serves as a helper strand which closes S1 and S2 through hybridization to facilitate the junction of nicks by T4 DNA ligase. In order to study the influence between hybridization effect and ligase efficiency, AGE was run to explore the optimized hybridization and ligation condition (Fig. 3C). The working temperatures of hybridization and ligation for the samples from lane 2 to 7 are 4 °C and 4 °C, 4 °C and 22 °C, 4 °C and room temperature, room temperature and 4 °C, room temperature and 22 °C, room temperature and room temperature, respectively. Since ligation produces a significant shift in DNA electrophoretic mobility, the bands with lower mobility appear in the lane 2, 3, 5 and 6 (the bands of lane 4 and 7 are failed for ligation). A relatively brighter band was observed in lane 5, reflecting the relatively better condition for ligation, that is, hybridization in room temperature and ligation in 4 °C overnight. Besides, hybridization in room temperature and ligation in 22 °C for 1 h in lane 6 also played a good effect next to lane 5. At the same time, the effect of T4 DNA ligase was also investigated by AGE. As shown in Fig. 3D, increasing concentrations of T4 DNA ligase greatly promoted the yield of this reaction. It can be seen that the higher the concentration of ligase, the brighter the band. It is probable that addition of enough ligase may guarantee the ligase could efficiently repair DNA nicks.
Footnotes |
† Electronic supplementary information (ESI) available: Experimental details and results. See DOI: 10.1039/c4ra11100a |
‡ These authors contributed equally to this work. |
This journal is © The Royal Society of Chemistry 2014 |