Principle of the sensor detection
In this paper, a label-free colorimetric biosensor based on non-specific adsorption of HCR and AuNPs was designed. According to the p53 gene, two hairpin probes H1 and H2 without any modification were designed (as in Scheme 1). The sticky end of the hairpin probe can be adsorbed on the surface of AuNPs through the interaction of positive and negative charges between its exposed nitrogen-containing bases and AuNPs [11]. In the presence of the p53 gene, the hybridization chain reaction was initiated. The p53 gene paired complementarily with the base at the 5' end of H1, causing the opening of probe H1. The newly exposed sticky end of H1 paired complementarily with the base at the 5' end of H2, leading to the opening of hairpin H2 and exposing its sticky end. The newly exposed sticky end of H2 had the same base sequence as the p53 gene, allowing it to repeat the chain reaction. This process resulted in the formation of long double-stranded DNA that could not be adsorbed onto the surface of AuNPs [12]. At this point, a NaCl solution was added to the solution. With increasing NaCl concentration, the Na+ ions neutralized the negative charge on the surface of AuNPs, leading to the continuous aggregation of AuNPs. As a result, the color of the solution changed from red to blue- violet. In the absence of p53 gene, the hairpin probes maintained their hairpin structures, and their sticky ends adsorbed onto the surface of AuNPs. The AuNPs adsorbed with hairpin probes remained dispersed in the salt solution, and the solution retained its red color.
The hairpin probe consisted of the sticky end, the stem and the loop, in order to meet the experimental requirements, H1 was composed of sticky ends of 12 bases, stems of 12 base pairs and loops of 10 bases, H2 was composed of sticky ends of 10 bases, stems of 13 base pairs and loops of 10 bases. They can be adsorbed on the surface of AuNPs through the sticky end in the absence of p53 gene, and there was no self-hybridization between H1 and H2 when they coexisted. In the presence of p53 gene, the p53 gene paired complementarily with the base at the 5’ end of H1, thereby opening probe H1. The newly exposed sticky end of probe H1 at the 5’end paired complementarily with the base at the 5’ end of probe H2, which then opened hairpin H2 to expose the sticky end on H2. The newly exposed sticky end of H2 had the same base sequence as the p53 gene and can repeat the above chain reaction instead [13]. Although the final long double-helix structure also had free sticky ends, the total number of probes H1 and H2 was greatly reduced compared to the initial solution, thus causing the salt-induced aggregation and the color change from red to blue-violet.
Feasibility demonstration of the method
In order to verify whether the p53 gene can induce the probe H1, H2 for HCR, the samples of H1, H2, H1-p53, H2-p53 and H1-H2-p53 were tested by gel electrophoresis and the results were shown in Fig. 1a. Both H1 and H2 were hairpin structures and appeared as bands on the lanes. When H1 and H2 were coexisting in the solution, they remained as bands on the lanes, so they would not hybridize by themselves. When p53 was present in the solution, the lanes appeared a smear, which can be proved that p53 induced H1 and H2 to produce a lot of long double helix structures. It should be noted that gel electrophoresis did not verify whether p53 induces HCR in H1, H2 according to the steps [14]. To verify the scientific validity of p53 induced HCR in H1, H2, we measured the fluorescence spectra of H1, H2, H1- p53, H2-p53 and other samples with Hoechst dye. Hoechst dye was a double-stranded DNA fluorescent dye that bound to DNA at the minor groove of the adenine and thymine enriched region with a fluorescence excitation wavelength at 480 nm, and the dye is added to the solution before detection [15]. The change in the number of base pairs in the solution would lead to a change in fluorescence intensity, which would determine whether a reaction had occurred, and the results were shown in Fig. 1b. p53 gene is a single-stranded DNA, so the fluorescence intensity of the solution did not increase. Base pairs of H1 and H2 caused an increase in fluorescence intensity. When H1 was mixed with the p53 gene, the fluorescence intensity at 480 nm changed, indicating that the p53 gene reacted with H1. When H2 was mixed with p53 gene, the fluorescence intensity did not change, indicating that p53 gene could not react with H2. When the solution was H1-H2-p53-Hoechst mixture, the fluorescence intensity at 480 nm increased sharply, and HCR occurred at this time. We used gel electrophoresis and fluorescence spectroscopy to demonstrate that the principle of p53 gene induced H1 and H2 to occur HCR were consistent with the design. Figure 1c showed that the lower the concentration of p53 in the same reaction time (one hour) the longer the double helix structure generated, and the more probes H1, H2 that did not participate in the reaction, the remaining H1, H2 would protect the AuNPs. After giving sufficient reaction time (Fig. 1d), H1 and H2 were eventually consumed and the long double helix structure increased significantly with decreasing p53 concentration, this result is similar to other literatures.
In addition, the samples H1-H2-AuNPs-Nacl and p53-H1-H2-AuNPs-Nacl mixed solutions were also measured by UV-Vis absorption spectrometry. The results were shown in Fig. 2E. The UV-Vis absorption spectrum of H1-H2-AuNPs-Nacl showed a clear absorption peak at about 520 nm, indicating that the probes H1, H2 played a good protective effect on AuNPs, and Na + could not make AuNPs aggregation and an adsorption peak of p53-H1-H2-AuNPs-Nacl at 520 nm decreased. The color of the solution changed from red to purple at this time (Fig. 1e, inset) [16]. The degree of aggregation of AuNPs can be studied more visually by TEM. Figure 1f showed that the probes H1, H2 adsorbed on the surface of AuNPs, and at this time the AuNPs were dispersed by adding NaCl solution. Figure 1G showed that after adding a small amount of p53(20nM), part of the probe underwent HCR, at which time NaCl induced partial aggregation of AuNPs. As shown in Fig. 1H, the higher concentration of p53(200nM) induced a large number of probes H1 and H2 formed a double helix structure that could not be adsorbed on the surface of AuNPs, and NaCl induced AuNPs to be completely aggregated. These experimental results verified the feasibility of p53 causing changes in absorbance or color of the H1-H2-AuNPs-Nacl mixed solutions.
Optimization of experimental conditions
The probes H1 and H2 are adsorbed on the surface of the gold nanoparticles through the nitrogen and oxygen atoms in the bases. (Supplementary Information Figure S1, S2) [17, 18]. The adsorption capacity of the probe is highly dependent on its own concentration and the reaction time with nanogold. In order to optimize the concentration of H1 and H2, the volume and concentration of AuNPs were fixed first, and the concentration of H1, H2 was changed. The sample H1-H2-AuNPs mixed solution was detected by UV spectrophotometer, the results were shown in Fig. 2a (A0 indicates the absorbance of the sample solution at 520 nm, A1 indicates the absorbance of the sample solution at 520 nm after the addition of NaCl), (A0-A1)/A0 decreased with the increase of H1, H2 concentration. The smaller the value of (A0-A1)/A0, the less AuNPs gathered after adding NaCl, and the more probes adsorbed on the surface of AuNPs, which proved that the adsorption capacity of probes H1, H2 increased with the increase of their own concentration. When the concentration was 350nM, the ordinate stopped decreasing, at which time most of the AuNPs in the solution were covered by H1 and H2. When the probe concentration reaches 437nM, the ordinate rises, which may be due to too high concentrations of H1 and H2 leads to a decrease in their adsorption rates [19], so the probe with a concentration of 350nM was chosen for the experiment. The optimized probe concentration was chosen, and the adsorption time of the probes with AuNPs was changed. As shown in Fig. 2b, the (A0-A1)/A0 of the sample H1-H2-AuNPs decreased with the increase of adsorption time, which indicated that the adsorption effect increased with the rise of adsorption time, and the ordinate tended to level off after 65 minutes, and a longer adsorption time would not cause significant changes in the ordinate. Therefore, 65 min was chosen as the adsorption time of the probe.
The designed probes H1 and H2 were adsorbed on the nanogold surface through the sticky end. It was necessary to adjust the sodium ion concentration to shield the negative charge on the DNA phosphate backbone allowing the sticky end to be closer to the nanogold, so that as many probes as possible could be adsorbed on the nanogold surface to achieve high sensitivity of the sensor [20]. For the quantitative detection of the p53 gene, sodium ions were also required to aggregate the nanogold and to analyze the experimental results by the naked eye or by UV spectrophotometer. If the concentration of Na+ was added too low, it would not cause the AuNPs which were not protected by the probe to aggregate, while the high concentration of Na+ tended to denature the AuNPs which would result in false positive result [21]. Therefore, the sodium ion concentration in the solution needed to be optimized both before and after the addition of the substance to be measured in order to improve the sensitivity.
Figure 2c showed the sodium ion concentration optimized to make the probe adsorbed on the nanogold surface. The A520/A620 ratio increased with increasing Na+ concentration in the range of 0 to 28mM, indicating that the adsorption capacity of H1 and H2 probes increased with the concentration of Na+. However, when the Na+ concentration was too high, it induced the aggregation of nanogold particles in solution, causing a decrease in A520/A620. Therefore, the concentration of Na+ used for the adsorption of probes H1, H2 was 28nM. Figure 2d illustrated the Na+ concentration required for the aggregation of nanogold after HCR. To avoid false positives, the samples of p53-H1-H2-AuNPs and H1-H2-AuNPs were tested. The ordinate increased with increasing Na+ concentration in the p53-H1-H2-AuNPs sample, indicating that higher Na + concentrations resulted in more aggregated AuNPs. For the H1-H2-AuNPs sample, the ordinate hardly increased when the Na+ concentration was between 35-70mM, suggesting that the AuNPs protected by the probe did not aggregate. However, the ordinate increased with increasing Na+ concentration when it exceeded 70 mM, indicating denaturation of AuNPs due to the high Na+ concentration.
Therefore, a Na+ concentration of 70 mM was chosen for the aggregation of AuNPs in the experiment. The aggregation of AuNPs by Na + is not an instantaneous process, so the optimal time required for complete aggregation needed to be determined. Figure 2e demonstrated that the ordinate of the p53-H1-H2-AuNPs sample increased with the placement time after adding NaCl, stabilizing between 35 to 125 minutes. This indicated that the NaCl solution could fully aggregate the unprotected AuNPs within 35 to 125 minutes. Thus, 35 minutes was chosen as the required time for complete aggregation of AuNPs by the NaCl solution.
The hybridization chain reaction is the key to this experiment and the change in DNA conformation is influenced by pH, temperature and reaction time. Adenine and cytosine will be protonated to carry a positive charge around pH 3 [22]. Although the phosphate backbone of DNA remains negatively charged, the total negative charge density of DNA decreases, which contributes to the occurrence of HCR. It was found that when the pH was below 5 or above 9, the double-stranded structure of DNA started to become unstable and the long double helix structure generated by HCR would be disrupted, resulting in the inability to amplify the p53 gene. Therefore, this experiment was all performed at a pH of 7.4. Although temperature has a great influence on the structure of both HCR and DNA hairpin, 37oC was used as the experimental temperature in order to simplify the experimental operation and to be more consistent with the human environment. Therefore, only the reaction time was optimized, and the results were shown in Fig. 5f, (A0-A1)/A0 of the sample p53-H1-H2-AuNPs increased with the increase of reaction time, and the more (A0-A1)/A0 rose indicated that the more AuNPs aggregated after the addition of NaCl, thus proving that the longer reaction time was, the more double-stranded structure HCR produced. The ordinate varied less from 20 to 26 hours and 20 hours was chosen as the reaction time for the experiment.
Detection limit of the p53 gene
Under the above optimal conditions, different concentrations of p53 (200, 100, 40, 20, 8, 4, 2, 1, 0.5 and 0.2nM) were set to explore the detection limits of the sensor, as shown in Fig. 3a, the absorption peak at 520 nm gradually decreased and the color of the solution gradually changed from red to blue-violet as the concentration of p53 gradually increased. The relationship between absorbance (A0-A1)/A0 and p53 concentration is illustrated in Fig. 3b. The detection range of the sensor is 0.2nM ~ 200nM, when the concentration of DNA was 0.2–5nM, there was a good linear relationship. The linear regression equation was y = 0.1022x + 0.00664(R2 = 0.9916), and the sensor has a detection limit of 0.2nM using UV spectrophotometer and 2nM with the naked eye, which is superior to other HCR-based colorimetric assays and less complex than sensors that were known to detect the p53 gene. (Supplementary Information Table 1 and Table 2) 1–5,7,20–26.
Detection specificity of the p53 gene
In order to verify the specificity of the sensor, four different target DNAs (200nM) were selected as single mutation, double mutation and two random DNA sequences. As shown in Fig. 4a, only the ordinate of the p53 gene was significantly decreased, so mutated or random DNA hardly induces H1, H2 triggers HCR, and this selectivity can be observed by the naked eye. As shown in Fig. 4b, the solution containing the p53 gene was blue-violet. The solutions for the rest of the target DNA are red. The experimental results are sufficient to prove that the sensor has good specificity for p53
Real sample analysis and recovery determination
In order to evaluate the usefulness of the sensor, 10% normal human serum containing different concentrations (0.2nM, 20nM and 200nM) of p53 was tested and the spiked recovery experiments were performed. The results are shown in Table 3, with spiked amounts of 0.23nM, 23.4nM and 202nM. The recoveries were 115%, 117% and 101%, respectively, indicating that this method is capable of detecting the P53 gene in real samples and has good
Table 1
Results of p53 gene assay in human serum
NO.
|
Added
(nmol/L)
|
Found
(nmol/L)
|
Recovery
(%)
|
RSD
(%, n = 3)
|
1
|
0.2
|
0.23
|
115
|
5.43
|
2
|
20
|
23.4
|
107
|
2.5
|
3
|
200
|
202
|
101
|
2.81
|