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A visual, label-free telomerase activity monitor via enzymatic etching of gold nanorods Haitang Yang, Anran Liu, Min Wei, Yuanjian Liu, Bingjing Lv, Wei Wei, Yuanjian Zhang, and Songqin Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02608 • Publication Date (Web): 24 Oct 2017 Downloaded from http://pubs.acs.org on October 25, 2017

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Analytical Chemistry

A visual, label-free telomerase activity monitor via enzymatic etching of gold nanorods Haitang Yang,#† Anran Liu,# † Min Wei,‡ Yuanjian Liu,† Bingjing Lv,† Wei Wei,*† Yuanjian Zhang,† Songqin Liu*† †

Jiangsu Engineering Laboratory of Smart Carbon-Rich Materials and Device,

Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, School of Chemistry and Chemical Engineering, Southeast University, Nanjing, 211189, China ‡

College of Food Science and Technology, Henan University of Technology,

Zhengzhou, 450001, China *

Corresponding author. Tel.: 86-25-52090613; Fax: 86-25-52090618.

#

Contributed equally to this work

E-mail address: [email protected] (W. Wei) [email protected] (S.Q. Liu)

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ACS Paragon Plus Environment


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ABSTRACT Early diagnosis and life-long surveillance are clinically important to improve the long-term survival of cancer patients. Telomerase activity is a valuable biomarker for cancer diagnosis but its measurement often used complex label procedures. Herein, we designed a novel, simple, visual and label-free method for telomerase detection by using enzymatic etching of gold nanorods (GNRs). Firstly, repeating (TTAGGG)x sequences were extented on telomerase substrate (TS) primer. It formed G-quadruplex under the help of Hemin and K+. Secondly, the obtained horseradish peroxidase mimicking hemin/G-quadruplex catalyzed the H2O2-mediated etching of GNRs to the short GNRs, even to gold nanoparticles (GNPs), generating a series of distinct color changes due to their plasmon-related optical response. Thus, this enzymatic reaction can be easily coupled to telomerase activity, allowing for the detection of telomerase activity based on vivid colors. This can be differentiated sensitively by naked eyes because human eyes are more sensitive to color variations rather than the optical density variations. As a result, telomerase activity can be quantitatively detected ranging from 200 to 15 000 HeLa cells mL-1. The detection limit was 90 HeLa cells mL-1 (S/N = 3). Importantly, the application of this method in bladder cancer samples was in agreement with the clinical results. Thus, this method was considerably suitable for point-of-care diagnostics in resource-constrained regions because of the easy readout of results without the use of sophisticated apparatus.

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INTRODUCTION Telomerase is responsible for maintaining the telomere length in cells. It is well known that telomerase is overexpressed in over 85% malignant tumor cells, while it is not expressed in almost all normal tissues. So, telomerase has potential to be used as a specific tumor marker. Consequently, accurate and efficient quantification of the telomerase is highly essential to pathologic diagnostics and therapeutics.1 The most commonly applied method is based on telomeric repeat amplification protocol (TRAPs).2,3 Recently, more and more PCR-free techniques were developed including colorimetric method,4,5 fluorescence,6-8 electrochemical,9,10 circular dichroism (CD) spectroscopy11 and surface-enhanced Raman scattering (SERS).12,13 Colorimetric methods are attractive due to their direct visual detection. Recently, our group has developed a colorimetric telomerase activity detection method based hemin-graphene nanomaterial.5 When telomerase was added, the color change was from colorless to dark blue through catalysis of 3,3',5,5'-tetramethylbenzidine (TMB). In addition, Freeman et al investigated the optical analysis of telomerase based on DNAzyme-like activity of hemin/telomeric G-quadruplexes. The obtained catalytic hemin/G-quadruplex units catalyzed the oxidation of TMB by H2O2. The oxidation of TMB to TMB.+ provided the readout signal for the telomerase activity. This process provided a colorimetric means to detect telomerase originating from 200 cells µL−1.14 Wang et al. reported the detection of human telomerase activity with primer-modified gold Nanoparticles (GNPs). The mechanism was based on the elongated primers conjugated to the GNPs surface. In the detection process, a rapid red-to-blue color 3



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change was correlated with the telomerase activity. The developed simple and sensitive colorimetric assay through GNPs aggregate could measure telomerase activity down to 1 HeLa cell µL−1.15 Despite this progress, the accuracy of visual inspection is poor due to the limited color display. It is well known that, human eyes are more sensitive to color variations (with spectral peak shift) rather than the optical density variations (no spectral peak shift).16 Therefore multicolor sensors which can be used in the detection of telomerase activity are still needed. As one of the most commonly used quasi-one-dimensional material, gold nanorods (GNRs) has been employed as a highly sensitive platform to investigate biologically important molecules through variations in their length.17 The most intriguing property of GNRs was their surface plasmon resonances (SPR). Owing to the anisotropic shape of gold nanorods, there were two plasmon modes corresponding to their width and length known as the transverse (TSPR) and longitudinal plasmon bands (LSPR).18 The TSPR locates at just above 500 nm while the LSPR varies widely according to the nanorods’ aspect ratio (AR, length divided by width). The varied AR gives rise to the vivid colors of their colloidal solutions.19-21 Under the inspiration by all of these fascinating characteristics, tremendous research progress had been made on studying the application of GNRs for sensing and detection of various analytes in biological and other systems during the past few years.22,23 For example, Ma et al. reported some methods using GNRs as colorful chromogenic substrates for semi-quantitative detection of nucleic acids, proteins, and small molecules with the naked eye.24 Saa et al. investigated the effects of horseradish 4


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peroxidase (HRP) on the enzymatic anisotropic etching of GNRs. They coupled HRP to glucose oxidase (GOx), which generated H2O2 from glucose oxidation, then provided a novel method for glucose detection, enabling the development of colorimetric glucose biosensors.25 These methods allow us to visually quantify the concentration of various target molecules with the naked eye, and the obtained results were highly consistent with those techniques that were tested by the advanced detection equipment. Herein, we take advantage of HRP mimicking hemin/G-quadruplex catalyze the H2O2-mediated etching of GNRs to fabricate a multicolor sensing strategy that can be used for telomerase activity detection. Under the catalysis of the obtained hemin/G-quadruplex DNAzyme, H2O2 decomposed into hydroxyl radicals with strong oxidizability, accelerating the GNRs etching.25 The catalytic reaction is a cyclic process consisting of twice single-electron transfer.26-30 During this reaction cycle, GNRs were shortened gradually and eventually turned into the Au(III) state. The color change companied with the GNRs etching was from orange-red to pink, causing a blue shift in UV-vis spectra. Specifically, we defined the brown GNRs (LSPR peak values at 841 nm) probe as origin, which represented urine extracts with inactive telomerase and implied normal individuals. The forward direction was corresponding to the detection of a relatively high concentration of active telomerase, where LSPR blue shift and color change of GNRs occurred obviously, predicting bladder cancer were suffered. It indicated that the method could be used to detect telomerase activity by naked eye. When it was used to detect telomerase in urine samples, high accuracy and reproducibility were obtained. Compared with previously reported colorimetric method, this method based on the LSPR shift of GNRs was more sensitive, avoided 5



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the complex label procedures and needed no chromogenic reagent. EXPERIMENTAL SECTION Preparation of GNRs. GNRs were prepared according to a simple one-pot method using hydroquinone as the reducing agent and NaBH4 as the initiating reactant. 21 In brief, the growth solution, HAuCl4 (48.56 mM), silver nitrate (0.02 M), and hydroquinone (0.2 M) were first sequentially added into 71.0 mL of CTAB (0.1 M) solution under slight shaking, followed by standing for 5 min. Then, a freshly prepared, ice-cold 0.5 mM NaBH4 solution (2.6 mL) was then injected into the mixture solution, followed by standing for over 12 h (final reactant concentrations are given in the figure S2A). The GNRs with different aspects ratios were synthesized by changing the amount of AgNO3. The obtained GNRs were centrifuged twice at 16 000 g for 10 min to remove excess CTAB. The obtained soft sediment was re-dispersed in ultrapure water and kept at room temperature. The colloid was found to be stable for at least six months. Its concentration was estimated by Lambert-Beer Law.31 Telomerase Extension and Inhibition Reaction. Cell cultivation and telomerase extraction were prepared according to our previous literature.32 After extraction, extension reaction buffer containing 63 mM K+, 1.0 mM dNTPs, 5 µM telomerase substrate (TS) primer were added with telomerase solution which contained specified number of HeLa cells. After incubation at 37 °C for 60 min, the formed G-quadruplexes were treated with 2 × 10-6 M hemin for 120 min at room temperature. To evaluate the inhibitors of BIBR 1532 or curcumin, various concentrations of them were incubated with 9 000 cells mL-1 HeLa cells respectively. Heated HeLa cells 6


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(95 °C, 5min) were used for control experiments. Colorimetric Detection. For the detection procedure, 5 µL of the telomerase extension products were added to 995 µL of citrate buffer (20 mM, pH = 3.0) containing GNRs and H2O2. After rapid inversion, the mixture solution was gently mixed. After 60 min, their absorbances were recorded. RESULTS AND DISCUSSION Sensing Mechanism of Enzyme-Induced Colorimetric Assay. Here, we represent the mechanism of enzyme-induced GNRs etching in Scheme 1. Firstly, in the presence of different amounts of active telomerase, the extension repeated units (TTAGGG)x were extended. Then, it folded into a number of multiple telomeric hemin/G-quadruplexes under the help of K+ and hemin.32 The obtained hemin/G-quadruplex DNAzyme catalyzed the H2O2-mediated oxidation of GNRs, resulting in color change of GNRs solution. The color of the solution varied from orange-red to pink corresponding to the active telomerase concentration. The effects of H2O2 on GNRs etching were first investigated (Figure 1A). It was found that when GNRs were directly oxidized by 60 mM H2O2 for 240 min, the LSPR band of GNRs had a blue shift (∆W) at about 8 nm. This indicated that the sole addition of H2O2 was unable to etch GNRs efficiently. Next, we investigated the accelerating effect of hemin/G-quadruplexes on GNRs etching (Figure 1B). Compared with the initial GNRs/H2O2 spectrum (Figure 1B, curve a), the spectrum of GNRs/H2O2 solution in the presence of TS primer (curve b) or G-quadruplexes (curve c) elongated by telomerase were almost as same as that for the initial GNRs/H2O2, 7



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indicating that TS primer and the formed G-quadruplexes had no catalytic activity. GNRs/H2O2 solution containing hemin (curve d) induced a small blue shift due to the weak catalysis of H2O2.28 When Hela cells/H2O2/hemin was used to etching GNRs (curve e), a little blue shift happened, but it is much smaller when compared to the same solution but incubated with the primer (curve f). This proved that the blue shift was induced by primer extented telomere DNA-hemin complex rather than other G-quadruplex DNA. The concomitant incubation of formed telomere G-quadruplexes and hemin with the GNRs/H2O2 led to a significant blue shift (∆W =128 nm), which indicated that G-quadruplex enhanced the catalytic activity of hemin greatly and telomere DNA is the main existence of G-quadruplex structure. Accordingly, the color of GNRs changed from orange-red to grey. Figure 1C showed the etching effects of H2O2 on GNRs etching in the presence of hemin/G-quadruplex DNAzyme. When compared with Figure 1A, it was obvious that the etching rate under the catalysis of the hemin/G-quadruplex DNAzyme was much faster than that in the presence of only H2O2. Thus, telomerase was very important to the etching of GNRs and its activity had great impacts on the color of GNRs. Figure 1D showed the corresponding LSPR shift of the GNRs with etching time from 0 to 120 min. 60 mM H2O2 and 9 000 HeLa cells mL-1 were used. Characteristic and Optimization of GNRs. GNRs with different ARs were prepared according to one-pot or seed mediated growth method.21,33,34 AR was an important parameter and should be strictly controlled in order to obtain best sensitivity and accuracy. We designed an experiment to compare the behaviors of three kinds of 8


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initial AR of GNRs. Considering that their initial LSPR peak values at 702, 779, and 841 nm, they were named as GNRs-702, GNRs-779, and GNRs-841, respectively. As shown in Figure S1, the LSPR shift of GNRs-841 was much faster than that of the others. Based on the sensing mechanism, GNRs with higher aspect ratio should work better.35,36 Because in this etching process, the AR of GNRs decreased and finally the GNRs became GNPs. Thus, the larger aspect ratio has more room to decrease, providing broader detectable linear range. The experiment results of Figure S1 were consistent with the sensing mechanism. Thus GNRs-841 with an AR of about 4.9 were used in the following studies. To characterize the morphologies of the obtained GNRs and the enzymatic etching process of GNRs, TEM images of GNRs samples collected at various stages of oxidation were showed in Figure 2. Figure 2A showed that the initial GNRs had a mean length of 50.4 nm and a mean width of 10.3 nm. The GNRs became shorter and finally transformed into the spherical particles with the increasing incubation time under the catalysis of hemin/G-quadruplex (0 to 90 min) (Figure 2B, C). Inset in Figure 2A,B,C showed the distinct different colors of GNRs, which was the base to construct a novel multicolor sensor for telomerase activity detection. Figure 2D showed the spectra of corresponding GNRs, where the LSPR band of GNRs had obviously blue shift (Figure 2D). These results confirmed the ability of hemin/G-quadruplex to facilitate the H2O2-promoted etching of GNRs. Moreover, the distributions of aspect ratios and their mean values analyzed from the corresponding TEM images were also studied (Figure S2 B,C,D), a decrease of the mean aspect ratio 9



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from 4.9 to 1.3 was observed in 90 min oxidation time, which is in accord with the blue shift of the LSPR band of GNRs. Analytical Performance for Colorimetric Detection of Telomerase Activity. Before the analytical performance of telomerase activity, we investigated the effects of H2O2 on the time-dependent DNA enzymatic etching of GNRs. Figure S3A showed the LSPR shift (∆W) of the GNRs as the function of oxidation time with varied concentration of H2O2. It was found that the etching rate of the GNRs was increased with the increased concentration of H2O2 (Figure S3 B). The higher concentration of H2O2 led to higher etching rate (Figure S3 A and B). It should be noted that the concentration of H2O2 have great impact on the detection limit and the linear range. When the higher concentration of H2O2 was used, the detection limit became lower because the response is very sensitive and the linear range became narrower due to GNRs would be quickly etched to GNPs. In order to obtain the lower detection limit and the reasonable linear range, 60 mM H2O2 were selected. As shown in Figure 3A, the color variations and corresponding LSPR shifts of GNRs etching with the increasing concentration of telomerase were recorded. When the telomerase concentration increased, the solution colors gradually transferred from orange-red to brown, to light grey, to green, to blue, to purple and finally to pink during this period. It should be noted that in the presence of high concentration of telomerase, GNRs were completely etched to GNPs and LSPR band disappeared. As can be seen in Figure 3B, C, the ∆W showed a proportional relationship with varied HeLa cells concentrations within the range from 200 HeLa cells mL-1 to 1 5000 HeLa cells mL-1 10


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(∆W =0.01398 C (cells mL-1) + 7.8475, R2 = 0.9961 (1) ), the limit of detection (LOD) was 90 HeLa cells mL-1 (based on the calculation of the average signal of the blank plus three times of the standard deviation). This result is acceptable in comparison to previously reported methods for telomerase detection (Table S1). It was more sensitive than most reported colorimetric methods. Especially, it is more sensitive than that obtained via G-quadruplex/hemin controlled aggregation of GNPs.37 Performance of the Strategy in Various Cell Lines. The practicality and accuracy of this method were investigated by using three kinds of other cells lines. As shown in Figure 4A, it suggested this proposed method are effective in various cancer cell types, especially for the HeLa cells and A549 cells. Such observation was in good agreement with previously reported results.5,38 However, when the cancer cells were heated at 95 °C, telomerase in cancer cells was inactivated, The primer cannot be extented and without quadruplex was formed to catalyze the etching of GNRs. As a result, negligible LSPR shift was observed, this indicated the specificity of the present assays. Selectivity of the strategy. BSA and Phi29 were used as controls to evaluate the selectivity of the strategy. As shown in Figure 4B, LSPR shift increased significantly when the reaction solution was spiked with HeLa cells. Whereas there was little signal change when heated HeLa cells, BSA and Phi29 were added. Such a comparison suggested the high selectivity and the great potential of this method for sensitive colorimetric detection for the target telomerase activity. Thus, the established strategy was suitable for selective detection of telomerase activity. 11



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Evaluation of Inhibition on Telomerase Activity. Substantial data suggested that telomerase was reactivated in most of tumorigenesis, but inhibited in a majority of normal somatic cells.39 Thus, it was significant to investigate the inhibition of telomerase. Two types of effective inhibitor including BIBR 1532 and curcumin were used. The inhibition efficiency (%) was given by the following formula (2): Inhibition (%) =

∆ ∆ ∆ ∆

x 100%

(2)

Where ∆W1 meant the LSPR shift of GNRs without telomerase, ∆W2 meant the LSPR shift of GNRs treated with 9 000 HeLa cells/mL incubated with inhibitor, ∆W3 meant the LSPR shift of GNRs treated with 9 000 HeLa cells/mL without inhibition. As shown in Figure 5, telomerase activity gradually decreased when the concentration of inhibitor increased and finally reached a platform. IC50 were obtained as 256.4 ± 54 nM for BIBR and 18.9 ± 3.1 µM for curcumin by using the softerware of “IC50 calculator”. This was in agreement with the reported results.4,40 Thus, the proposed method had a potential application in discovery of new telomerase inhibitors. Thus, the proposed method had a potential application in discovery of new telomerase inhibitors. Clinical Applicability. Owning to the environmental and dietary exposures, bladder cancer (BC) became the second most common genitourinary malignant disease.41 In order to demonstrate the applicability and reliability of the proposed assay in clinical application, 11 early morning urinary samples provided by Nanjing General Hospital of Chinese People's Liberation Army were detected. Telomerase from human urine specimens were extracted in a typical experiment.32 Urine specimens were centrifuged, 12


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washed and then redispersed and incubated in lysis buffer. Finally, after the centrifugation at 12 000 rpm for 20 min, the obtained supernatant was used for telomerase extension and analysis. The extension products were added to citrate buffer containing GNRs and H2O2. After rapid inversion, the mixture solution was gently mixed. After 60 min, the color change and absorbance was recorded. According to the colorimetric sensing strategy for cancer diagnosis, 25 nm LSPR shift was proposed as threshold value to evaluate the bladder cancer development (obtained by the mean ∆W of 50 blank samples plus 3 times the standard deviation). As can be seen in Table 1, No.1, 2, 3, 5, 6, 7, 8 were from noncancer patients. Others in Table 1 were from bladder cancer samples. The color of GNRs probes in normal individuals, urinary lithangiuria or inflammation patients were still orange-red and the values of ∆W were less than threshold value, which can be further proved by its UV−vis spectrum (Figure S4). All bladder cancer samples had a large LSPR shift above threshold value and the severity level can be differentiated by the color of GNRs. These judgments agreed well with clinical analysis results. Therefore, this painless method can differentiate the cancer and noncancer urine specimens and it was promising for bladder cancer diagnosis. CONCLUSIONS In this paper, a simple, label-free colorimetric method was developed for monitoring

telomerase by

using

naked

eyes. Ultrahigh

HRP mimicking

hemin/G-quadruplex/H2O2 induced GNRs etching was considered as a signal output. GNRs etching could be triggered through the hydroxyl radicals from HRP mimicking 13



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hemin/G-quadruplex-catalyzed H2O2. Among them, GNRs were directly used without complex modification procedures, hemin/G-quadruplexes with high catalysis were elongated by telomerase themselves. Owing to the combined advantages of hemin/G-quadruplexes catalytic activity for H2O2 and multiple color responses (orange-red to pink) generated through GNRs etching, the proposed method showed a good linear ranged from 200 HeLa cells mL-1 to 15 000 HeLa cells mL-1 with a LOD of 90 HeLa cells mL-1, which was more sensitive than that obtained via G-quadruplex/hemin controlled aggregation of GNPs. The method had also been applied in several human urine samples. Results showed that telomerase activity was negative for normal and inflammation samples, while it was positive for bladder cancer samples. This assay possessed several appealing features, such as easy preparation, low cost, good specificity and high accuracy. Therefore, the assay showed its potential to be applied in biological analysis, which might be significant in disease diagnosis in the future. ASSOCIATED CONTENT Supporting Information Additional information is available free of charge via the Internet on the ACS Publications website. Comparison of the detection performance with various GNRs, TEM image of used GNRs, distributions of aspect ratios of used GNRs, detection performance with varied concentration of H2O2, the UV-vis spectra changes of GNRs for telomerase detection from urine samples and comparison of analytical performance of various methods for determination of telomerase activity. 14


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AUTHOR INFORMATION Corresponding Author *Phone:

86-25-52090613.

Fax:

86-25-52090618.

E-mail:

[email protected];

[email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (Nos. 21775019, 21475020, 21375014 and 21627806), the Fundamental Research Funds for the Central Universities and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (Grant Nos. 1107047002).

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Figure captions Scheme 1 Schematic illustration of colorimetric detection of telomerase activity via enzymatic etching of GNRs.

Figure 1 The UV-vis spectra change of GNRs in the absence (A, from 0 to 240 min) and presence (C, from 0 to 120 min) of hemin/G-quadruplex. (B) Control experiments of the enzyme-induced etching and colorimetric detection. a: GNRs, b: GNRs + TS primer, c: GNRs + G-quadruplex, d: GNRs + hemin, e: GNRs + hemin/Hela Cells, f: GNRs + hemin/G-quadruplex. The insets of (B) showed the photographs of the corresponding color of GNRs. (D) Corresponding LSPR shift of the GNRs with changing etching time. 60 mM H2O2 and 9 000 HeLa cells mL-1 were used.

Figure 2 TEM graphs of GNRs with etching time of 0 min (A), 65 min (B), and 90 min (C). The insets of (A), (B) and (C) showed the photograph of the corresponding GNRs. (D) Corresponding UV-vis spectra of GNRs with different etching time. 60 mM H2O2 and 9 000 HeLa cells mL-1 were used.

Figure 3 (A) Color change of the proposed sensor with the increasing concentrations of HeLa cells. From 1 to 15: 0, 200, 400, 600, 800, 1 000, 3 000, 5 000, 7 000, 9 000, 11 000, 12 000, 15 000, 19 000 and 22 000 HeLa cells mL-1. (B) UV-vis spectra for different concentrations of HeLa cell. From a to k: 0, 200, 1 000, 3 000, 5 000, 7 000, 9 000, 11 000, 13 000, 15 000 and 22 000 HeLa cells mL-1. (C) Linear relationship between ∆W of GNRs versus different concentration of HeLa cells in (B). 60 mM 20


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H2O2 was used. Etching time (t) = 60 min. Figure 4 (A) Telomerase activity detection for various cell lines. (B) Selectivity of the biosensor. 9 000 cells mL-1 were used for each cell line. Heated inactive HeLa cells were used as negative control. Error bars showed the standard deviation of three samples.

Figure 5 Inhibition effect of BIBR 1532 (A) and curcumin (B) on telomerase activity. 9 000 HeLa cells mL-1 were used. Error bars showed the standard deviation of three samples.

Table 1 Comparison of the results obtained by the proposed method and clinical diagnosis.

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Scheme 1

22


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Figure 1

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Figure 2

24


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Figure 3

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Figure 4

26


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Figure 5

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Table 1 Comparison of the Results Obtained by the Proposed Method and the Clinical Diagnosis α No.

α

Patient ID

clinical outcome

∆W

1

Water

-

12

2

Normal

Normal

12

3

1003295831

Vesical calculus

19

4

1008455348

Bladder cancer

175

5

1002693440

Kidney stone

18

6

1008455719

Kidney stone

15

7

1008461668

Kidney stone

18

8

1007144483

Inflammation

20

9

1007677148

Bladder cancer

130

10

1007922178

Bladder cancer

102

11

1007045669

Bladder cancer

166

12

1007924814

Bladder cancer

136

Clinical outcomes provided by Nanjing General Hospital of Chinese People's Liberation Army.

28


Colors

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For TOC only

29


A visual, label-free telomerase activity monitor via ... - ACS Publications

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