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Gold nanobipyramids as dual-functional substrates for in situ “turn on” analyzing intracellular telomerase activity based on target-triggered plasmon enhanced fluorescence Shenghao Xu, Liping Jiang, Yongyin Nie, Jun Wang, Haiming Li, Yuanyuan Liu, Wei Wang, Guiyun Xu, and Xiliang Luo ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b05447 • Publication Date (Web): 25 Jul 2018 Downloaded from http://pubs.acs.org on July 26, 2018

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ACS Applied Materials & Interfaces

Gold nanobipyramids as dual-functional substrates for in situ “turn on” analyzing intracellular telomerase activity based on targettriggered plasmon enhanced fluorescence †

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Shenghao Xu, Liping Jiang, Yongyin Nie, Jun Wang, Haiming Li, ǂ Yuanyuan Liu, Wei Wang, † † Guiyun Xu, and Xiliang Luo* † ǂ

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Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, P. R. China Qingdao Women and Children’s Hospital, Qingdao 266034, P. R. China ABSTRACT: Herein, we developed a novel plasmon enhanced fluorescence (PEF)-based telomerase-responsive nanoprobe for in situ fluorescence “turn on” visualization of telomerase activity in live cells. The as-prepared nanoprobe was composed of a nicked molecular beacon (which contains Cy5.5-labeled hairpin DNA sequences hybridized with telomerase primers) functionalized gold nanobipyramids (Au NBPs). Au NBPs were selected as both fluorescence resonance energy transfer (FRET) and PEF dual-functional substrates, while DNA was selected to be the precise spacer to manage the interval between the Au NBPs and Cy5.5. Based on this target-triggered PEF probe, optimal fluorescent enhancement can be obtained with 49 DNA bases, which was higher than gold nanorods (Au NRs). The proposed method accomplishes sensitive telomerase activity detection down to 23 HeLa cells with a dynamic range from 40 to 1200 HeLa cells. Based on this, in situ fluorescent imaging of telomerase activity in live cells and real-time analysis of the variation in intracellular telomerase activity can be achieved. Moreover, cancer cells and normal cells can also be successfully discriminated even in their cocultured mixtures, indicating promising potential in clinical diagnoses. Keywords: Plasmon enhanced fluorescence; Gold nanobipyramids; Telomerase activity; In situ imaging; Turn on

INTRODUCTION

gold nanoparticles for in situ detection of intracellular telomerase activity with a fluorescence “turn on” mode;15 quantification and real-time detection of telomerase activity in living cells could be achieved by its one-step incubation technique. Similarly, by using FITC-labeled hairpin DNA sequences hybridized with telomerase primers and gold nanoparticle, Gu’s group developed a telomerase-triggered molecular beacon for detection of telomerase activity in living cells as well as precise cancer treatment.16 Nevertheless, shortcomings such as photobleaching, low signal intensities of fluorescent dyes with low concentration are still major bottlenecks for further application in situ telomerase detection in live cells.17 Therefore, developing sensitive strategies to overcome the intrinsic defect of low signal intensity of fluorescent dyes for in situ analysis of telomerase activity in live cells is urgently needed.

Highly sensitive and selective detection of tumor-related biomarkers is of great significance for cancer diagnosis and pathogenesis.1 Telomerase, one of the most common biomarkers for managing cancer, is a ribonucleoprotein that can add the repetitive (TTAGGG)n sequences at the chromosome level, resulting in the limitless proliferation of tumor cells.2,3 Substantial studies demonstrate that highly expressed or reactivated telomerase activity exists in more than 90% of human cancer cells, while this activity is absent or extremely depressed in normal cells.4 Therefore, this differential expression makes telomerase a valuable indicator for cancer diagnoses.5 Until now, various techniques have been devised for telomerase activity analysis.6-14 Real-time telomere repeat amplification protocol (RT-TRAP) is the most popular method for telomerase activity detection. However, false negative results usually occur because of timeconsuming polymerase chain reaction (PCR) steps.6 Therefore, great efforts have been made to exploit PCR-free methods for telomerase activity detection to overcome this shortcoming, including chemiluminescence, electrochemistry and colorimetry.79 However, time-consuming procedures or failed in situ detection still limit the widespread application of these methods. Recently, as a powerful technique, fluorescence imaging with low interference for in situ detection of telomerase activity at cellular level has been reported. For example, Ju’s group reported a robust probe consisting of a Cy5 labeled nicked molecular beacon and

Plasmon-enhanced fluorescence (PEF)-based nanostructures are considered effective substrates to solve the inherent shortcomings of traditional fluorophores, because of their excellent capabilities of enhancing fluorescent signals and improving detection sensitivity.18,19 The fluorescence intensity of fluorophores can be forcefully strengthened due to the plasmoninduced electric fields when the excited-state fluorophores with a suitable absorption or emission wavelength are located adjacent to PEF substrates.20,21 So far, gold nanostructures with diverse morphologies, including gold nanorods (Au NRs), gold

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Scheme 1 Schematic illustration of the PEF-based telomeraseresponsive nanoprobe for “turn on” in situ analyzing telomerase activity in live cells.

nanoparticles(Au NPs) and gold nanobipyramids (Au NBPs), are used as PEF substrates for enhancing the fluorescence emission of fluorophores because of their superior biocompatibility and inoxidizability. 22-25 Among these, Au NBPs are expected to be more suitable PEF substrates than Au NRs because of their two sharper apexes, which are conducive to the severalfold larger local electric field enhancement based on the lightning-rod effect. 26 Nevertheless, to the best of our knowledge, utilizing Au NBPs as PEF substrates to enhance fluorescent signals for in situ analysis of telomerase activity in live cells has not been explored yet. In this work, a PEF-based telomerase-responsive nanoprobe for in situ “turn on” visualization of telomerase activity in live cells is examined and combined with the superiorities of Au NBPs as both FRET and PEF substrates. As shown in Scheme 1, the Au NBP was functionalized with a nicked molecular beacon (MB), which was composed of Cy5.5-labeled hairpin DNA sequences hybridized with telomerase primers (TSP). First, after conjugation of the 3’ end to the Au NBP by a Au-S bond, the Cy5.5 fluorescence from the 5’ label could be quenched due to fluorescence resonance energy transfer (FRET). Then, the existence of dNTPs and telomerase outstretched the primers, resulting in opening of the hairpin loop by inner chain substitution followed by the release the quenched Cy5.5 away from Au NBP, switching the fluorescent state from “off” to “on”. Meanwhile, the distance between Au NBP and Cy5.5 could be controlled by adjust the number of oligonucleotides bases to obtain the best PEF effect. Consequently, the fluorescence can be enhanced 10.4-fold by Au NBP, which is higher than that by Au NR (4.3-fold). Meanwhile, with the advantages of excellent specificity and intracellular stability for telomerase identification, this PEF-based telomerase-responsive nanoprobe was successfully used to specifically differentiate normal and tumor cells in situ based on the level of telomerase activity. Moreover, dynamically analyzing the reduction of telomerase activity by the treatment of anticancer drugs could also be achieved by using this PEF-based telomeraseresponsive probe, demonstrating feasible probability in cancer diagnostic and telomerase-targeted drug screening.

EXPERIMENTAL SECTION Chemicals. HAuCl4·3H2O, ascorbic acid, sodium borohydride, cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), sodium citrate, epigallocatechin gallate (EGCG) and silver nitrate were obtained from Sigma-Aldrich (USA). Telomerase ELISA kit was purchased from Innovation Beyond Limits (Germany). 3-(4,5-dimethylthiazol-2-yl)-2-diphenyltetrazolium bromide (MTT), DNase I endonuclease and dNTPs were all obtained from Takara Biotechnology Co. DNA sequences were synthesized in Sangon Biological Engineering Technology. Detailed DNA sequences are listed in Table S1. Instruments. JEM-2100 transmission electron microscope (TEM) with an accelerating voltage of 120 kV(Hitach, Japan) was used to achieve the morphologies of all samples. F-7000 fluorescence spectrometer (Hitach, Japan) was used to perform the fluorescence spectra. Nano-ZS Zetasizer ZEN3600 (Malvern, U.K.) was used to measure zeta potentials. Fluo ViewTMFV1000 (Olympus, Japan) was used to obtain the confocal images. Polyacrylamide gel electrophoresis was carried out by using DYCZ-24D electrophoresis apparatus. Bioimaging system (UVP, USA) was used to get the fluorescence images. Synthesis and purification of Au NBPs. For the synthesis: the Au NBPs were synthesized by a modified seed-mediated growth method. First, HAuCl4 ( 125 µL of 0.01 M), sodium citrate (250 µL, 0.01 M )and 9625 µL of deionized water were mixed and stirred for 10 min. Subsequently, freshly ice-cold NaBH4 (150 µL, 0.01 M) was injected to the mixture. After stirring for 20 min, the mixture was aged at 30 °C for 120 min to achieve the seed solution. Whereafter, HAuCl4 (5 mL, 0.01 M), AgNO3 (1 mL, 0.01 M), HCl (2 mL, 1 M) and ascorbic acid (0.8 mL, 0.1 M) were sequentially added to CTAB solution (100 mL, 0.1 M) to get the growth solution. At last, the rough products were obtained by adding 6 mL seed solution to the growth solution with standing for 12 h at 30 °C. For the purification: First, approximately 220 mL of the rough products were centrifuged for 30 min at 13000 rpm. Then, the obtained precipitate was redissolved into CTAC (160 mL, 0.08 M). Afterwards, the mixtures of ascorbic acid (15 mL, 0.1 M), the redissolved solutions and AgNO3 (50 mL, 0.01 M) were aged at 65 °C for 5 h and then centrifuged for 30 min at 13000 rpm. Then, the obtained precipitate was redissolved into CTAB solution (150 mL, 0.1 M) and keep tranquil at 30oC for 12 h. Thus, the black Au/Ag Nanorods at the bottom were redispersed in 100 mL of deionized water. Afterwards, 1.6 mL of 25% NH3·H2O and 0.8 mL of 30% H2O2 were injected to the above solution and maintained tranquil for 3 h with the color changing from green to brown. At last, the supernatant was centrifuged for 20 min at 12000 rpm and baptised with deionized water for thrice. Thus, the pured Au NBPs were obtained by redispersed the precipitate into 60 mL of deionized water. Preparation of nicked MB functionalized Au NBPs. HairpinDNA (10 µL, 10 µΜ) and TSP (10 µL, 100 µΜ) were added to 500 µL of Au NBPs solution and stirred at 25 oC for 8 h. Then,

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ACS Applied Materials & Interfaces purification. In addition, Figure 1C showed the color changes of one-to-one correspondence at every step.

after centrifuging at 12000 rpm for 10 min, the obtained precipitate was washed with PBS twice and finally redispersed in 500 µL of PBS. The excess Cy5-tagged hairpin-DNA in supernatant was collected for determining the amount of MB on each nanoprobe. The dispersity of the as-prepared probe characterized by TEM. 20 µL of the as-prepared probe was added to 0.5 mL of PBS, RPMI 1640, FBS and cell extracts, respectively. Then, the TEM images were taken after 2 h on a copper grid. Demonstration of telomerase-triggered fluorescence. dNTPs (5 µL, 10 mΜ) and telomerase (5 µL, 40 U/L) were added to 250 µL of Au NBPs solution. Subsequently, the fluorescence intensity was recorded after various incubation times.

Cytotoxicity and in situ intracellular telomerase activity fluorescent imaging. MTT assay was performed to investigate the cytotoxicity of PEF-based telomerase-responsive probe on HeLa cells. First, heLa cells were cultured under the conditions of 5% CO2 and 37 °C for 24 h. Aftersards, various amounts of PEF-based telomerase-responsive probe were injected and cultivated for 24 h. At last, cell viabilities against the nanoprobe were obtained by detecting the absorbance at 490 nm using enzyme-labeling instrument. For in situ cell imaging, HeLa and L-02 cells were co-cultured in confocal dish to adhere for 24 h in the presence of 10% fetal bovine serum, penicillin (100 µg/mL), roswell park memorial institute-1640 and streptomycin (100 µg/mL) under the conditions of 37oC in a humidified atmosphere containing 5% CO2. Afterwards, 20 µL10 mM dNTPs and 20 µL probe were added to the confocal dish and cultured at 5% CO2 and 37°C for 2 h, which were ready for confocal fluorescence imaging. Furthermore, hoechst 33342 (a nuclei stain fluorescent dye) was also used to clearly show the distinguishment. In details, 20 µL10 mM dNTPs and 20 µL probe were added to the confocal dish and cultured at 5% CO2 and 37°C for 1.5 h. Afterwards, 20 µL hoechst 33342 was added to the confocal dish and continued to culture for 0.5 h. At last, the confocal fluorescence images were obtained. In order to confirm the as-prepared probe was indeed endocytosed both by normal cells and cancer cells, the cells on the confocal dish were digested, and then the TEM image was taken after dropping it on a copper grid.

Figure 1. (A) Schematic illustration of the purification of Au NBPs. (B) TEM images of (B-a) Au NPs and Au NBPs; (B-b) Au/Ag NRs and Au/Ag NPs; (B-c) Au/Ag NRs ; (B-d) Au/Ag NPs; (B-e) Purified Au NBPs; (B-f) Purified Au NPs. (C) The color changes of one-to-one correspondence to every steps. It has been proved that the PEF would be optimal when a stronger spectral overlap existed between the absorption/emission of the dye and the plasmon band of metal nanostructures.18,29,30 In this study, Au NBPs with a maximum plasma at approximately 700 nm were obtained by tuning the volumes of the seed solution, to achieve the highest overlap with the absorption/emission spectra (λmax,abs = 686 nm, λmax,em= 704 nm) of Cy5.5 (Figure 2A). Then, after the AuNBPs were functionalized with the nicked MBs, the UV−vis spectrum revealed the characteristic peak of DNA at 260 nm (Figure 2B), and the obtained probe showed a more negative zeta potential than the AuNBPs (Figure 2C), indicating the successful conjugation of the negatively charged MBs to AuNBPs. The number of MBs assembled on each probe was determined to be approximately 75 (Figure S2, Supporting Information). 31

RESULTS AND DISCUSSION Characterization and construction of the PEF-based probe. The preparation and purification of the Au NBPs are shown in Figure 1A. The crude products were composed of Au NPs and Au NBPs (Figure 1B-a). For the purpose of obtaining pure Au NBPs, first, a mixture of Ag-coated Au NPs and Agcoated Au NRs was achieved by growing Ag on the crude products (Figure 1B-b). Then, the Ag-coated Au NRs (Figure 1B-c) and Ag-coated Au NPs (Figure 1B-d) were separated based on their diverse depletion interactions among the surfactant micelles. 27,28 Finally, the purified Au NBPs (Figure 1B-e) and Au NPs (Figure 1B-f) were obtained after etching the Ag on their surface by NH3·H2O and H2O2. The ultraviolet absorption spectrum (Figure S1) further demonstrated the successful

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Figure 2. (A) Absorption spectra (red line) and emission spectra (blue line) of Cy5.5, and the extinction spectra of Au NBPs (black line) with longitudinal plasmon resonance wavelength at 700 nm. (B) UV-vis spectra of the AuNBPs (red line) and the MB founctionalized AuNBPs (blue line). (C) Zeta-potentials of the Au NBPs and the MB founctionalized AuNBPs. (D) Native polypropylene acylammonia gel electrophoresis of (a) ladderDNA, (b) hairpin-DNA, (c) MB (the hybridization of TSP and hairpin-DNA), (d) MB incubated with cell extract and dNTPs for 60 min, (e) MB incubated with telomerase and dNTPs for 60 min.

Figure 3. (A) the fluorescence intensity ofthe probe with different DNA base numbers. (B) Fluorescence enhanced factor of the probe with different DNA base numbers. (C) Fluorescence spectra of the mixtures of the nanoprobe, 0.8 U/L telomerase and dNTPs (200 µM each) for 0, 20, 40, 60, 80, 100, 120 and 140 min. (D) Plots of fluorescence intensity against reaction time of the nanoprobe and dNTPs without (black line) and with (red line) of 0.8 U/L telomerase.

The mechanism of the telomerase-responsive fluorescence switch. The mechanism of conversion by telomerase-triggered TSP extension and hybridization was investigated by gel electrophoresis (Figure 2D). First, MB was obtained by hybridization of TSP and hairpin-DNA for 60 min, and the product was approximately 20 bp longer than hair-DNA (Figure 2D, lanes b and c), demonstrating the constitution of the MB. Then, as shown in Figure 2D, lanes d and e, the obtained MB was incubated with dNTPs and cell extract or telomerase for 60 min, and the corresponding products were approximate 6 bp longer than the MB, indicating the telomerase triggered TSP extension.

To emphasize the advantages of Au NBPs to be preferable PEF substrates, Au NRs (TEM image in Figure 4A) with an optimal plasma absorption peak at approximately 700 nm, which also overlapped strongly with the absorption/emission band of Cy5.5, were synthesized for comparison (Figure 4B). The distance between Cy5.5 and Au NRs was the same as that of Au NBPs, which was also regulated by using 49 DNA bases. As shown in Figure 4C, the enhanced fluorescence factor based on Au NRs (4.3-fold) was lower than that of Au NBPs (10.4-fold). Moreover, according to the finite-difference time-domain (FDTD) simulations, Au NBPs possess a stronger electric field than Au NRs, which gives a theoretical motivation for the stronger fluorescence signal (Figure 4D). Therefore, experimental and theoretical results both indicated that AuNPRs possessed excellent capability for PEF compared to AuNRs.

PEF study of the telomerase-responsive probe. It is worth noting that PEF is of great use when developing ultrasensitive fluorescence probes due to its ability to improve fluorescent intensity as a result of strong interactions between metal cores and fluorophores. 21 Meanwhile, fluorescence enhancement also depended on the distance between metal cores and fluorophores. In this study, the distance between Cy5.5 and Au NBPs was regulated by varying the DNA base numbers. It can be seen from Figure 3A that the fluorescent intensity of the nanoprobe depended on the DNA bases, and a maximum fluorescence enhancement factor of 10.4-fold was obtained with 49 DNA base numbers (Figure 3B). This result, further indicated that the fluorescence of the dye-metal structures was distance dependent. In addition, the incubation time was optimized to obtain the best response to telomerase. As shown in Figure 3C, with the incremental incubation time, the fluorescence intensity of the nanoprobe increased step by step and achieved saturation after 80 min, while no obvious change was observed without telomerase, indicating that telomerase triggered fluorescence conversion (Figure 3D).

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ACS Applied Materials & Interfaces triggered fluorescence restoration. Moreover, TEM images also demonstrated that the as-prepared probe possessed good dispersity in PBS, RPMI 1640, FBS and cell extracts (Figure S5). Subsequently, after adding dNTPs and the nanoprobe into HeLa cells, in situ telomerase activity fluorescence imaging was studied according to the fluorescence restoration. As shown in Figure 5A, due to the telomerase-triggered TSP extension product, the recovered fluorescence intensity in the cytoplasm increased step by step with the incremental incubation time and reached saturation at 90 min, indicating that telomerase was located in the cytoplasm. 32 Furthermore, TEM images (Figure S6) also confirmed that the as-prepared probe was indeed endocytosed both by normal cells and cancer cells. For real-time analysis of the alteration of telomerase activity in live cells and further validation that telomerase triggered the fluorescence restoration in tumor cells, HeLa cells with diverse concentrations of telomerase activity were acquired by preprocessing using diverse concentrations of EGCG (telomerase inhibitor). It can be seen from Figure 5B that the fluorescence intensity decreased step by step with increasing EGCG, further demonstrating that this telomerase-responsive nanoprobe was specific to telomerase activity. It can be concluded that this PEFbased telomerase-responsive nanoprobe can be used for reflecting telomerase activity in live cells, showing feasible potential in telomerase inhibits drugs screening. Moreover, to investigate the potential of the designed nanoprobe, in vitro analysis of telomerase analysis was also performed by using extracts from HeLa cells as the telomerase provenience. This nanoprobe was first mixed with diverse concentrations of HeLa cell extracts at 37°C for 1.5 h, and then the fluorescence spectra were collected. The recovered fluorescence intensity was increased step by step with the increase in the numbers of HeLa cells (Figure 6A). An excellent linear correlation exists over the range from 40 to 1200 HeLa cells, together with the detection limit (LOD) of 23 HeLa cells utilizing the 3σ/s method33 (Figure 6B), which is better than recently reported.34 The detection limit of this work was also compared with other methods for telomerase analysis (see Table S2). Comparatively, the detection limit of the present work is comparable to, or even better than those of previously reported approaches for telomerase detection. The telomerase activity in a single HeLa cell was approximated to be 2.9 × 10−11 U using a standard curve combining with the enzyme-linked immunosorbent assay kit analysis (Figure S7)

Figure 4. (A) TEM image of the Au NRs with plasma absorption peak approximate 700 nm. (B) Absorption spectra (red line) and emission (blue line) of Cy5.5 and absorption spectra of Au NRs (black line) with plasma absorption peak approximate 700 nm. (C) Fluorescence spectra of MB (black line), Au NRs@MB (red line) and Au NBPs@MB (blue line).(D) The computed electric field maps of Au NBPs and Au NRs at 700 nm, respectively. In situ telomerase activity fluorescent imaging in live cells.

Figure. 5 (A) Confocal fluorescent images of HeLa cells in the presence of 20 µL nanoprobe over time. (B) Confocal fluorescent images of HeLa cells in the presence of diverse concentrations of EGCG (0, 25, 50, 75 and 100 nM), and then incubated with 20 µL of nanoprobe for 1.5 h. Before the intracellular usage, the stability of the nanoprobe opposite to enzymatic cleavage taking DNase I was investigated as an example. As shown in Figure S3, contradistinguished with telomerase-triggered fluorescence restoration, the nanoprobe showed negligible fluorescence restoration in the presence of 2 µg of DNase I after incubation for 120 min, demonstrating excellent stability of the nanoprobe opposite to nuclease cleavage. Additionally, this probe also demonstrated excellent stability in PBS or RPMI 1640 and FBS (Figure S4), demonstrating that the culture medium showed negligible influence on telomerase

Figure 6. (A) Fluorescence emission spectra of the nanoprobe in response to telomerase activity with various numbers of HeLa cell

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extracts. (B) Plot of the relative fluorescence intensity versus the HeLa cell numbers.

providing a potential tool for clinical diagnosis. We expect the present work offers a new pathway for designing sensitive PEF probes for recognizing cancer-specific biomolecules.

Distinguishing normal cells from tumor cells. For the purpose of verifying the potential of the nanoprobe for in situ telomerase activity fluorescence imaging , HeLa cells and L-O2 cells were used for further investigation. As shown in Figure 7A and 7B, confocal images revealed that L-O2 (normal cells) exhibited lower telomerase activities than HeLa cells (cancer cells). Moreover, the capability of the probe for distinguishing tumor cell in the mixtures of normal cells and tumor cells was also investigated. It can be seen from Figure 7C that the fluorescence can be only detected in the HeLa cells, while no fluorescence can be detected in L-O2 cells. To clearly show the distinguishing features, Hoechst 33342 (a nuclei stain fluorescent dye) was used. As shown in Figure S8, blue fluorescence can be observed in both normal cells and cancer cells; however, red fluorescence can only be observed in cancer cells, demonstrating the feasible potential of the nanoprobe for distinguishing normal cells from cancer cells.

ASSOCIATED CONTENT Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Fax :+86-532-84022681

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors gratefully acknowledge the support from the National Nature Science Foundation of China (21675093, 21505081), the Doctoral Found of QUST (010022832) and the Taishan Scholar Program of Shandong Province, China (ts20110829).

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Figure 7. Confocal images of (A) HeLa cells, (B) L-O2, (C) cocultivation of HeLa cells and L-O2 cells after incubating with the nanoprobe and dNTPs for 90 min.

CONCLUSIONS In summary, we introduced a PEF-based telomerase-responsive nanoprobe for in situ “turn on” visualization of telomerase activity in live cells using Au NBPs as the dual functionalized substrates. By controlling the number of DNA bases to regulate the distance between dyes and Au NBPs, the fluorescence intensity can be enhanced up to 10.4-fold, which was higher than that achieved by using Au NRs. With advantages of good biocompatibility, high stability and satisfactory specificity, dynamic in situ telomerase activity fluorescence imaging in HeLa cells was obtained. Moreover, cancer cells and normal cells in their cocultured mixtures could still be well distinguished from each other,

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