Subscriber access provided by READING UNIV
Article
"Turn-on" fluorescent aptasensor based on AIEgen labeling for the localization of IFN-# in live cells Ke Ma, Fengli Zhang, Nima Sayyadi, Wenjie Chen, Ayad Anwer, Andrew Care, Bin Xu, Wenjing Tian, Ewa M. Goldys, and Guozhen Liu ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00720 • Publication Date (Web): 08 Jan 2018 Downloaded from http://pubs.acs.org on January 8, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Sensors is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Sensors
"Turn-on" fluorescent aptasensor based on AIEgen labeling for the localization of IFN-γ in live cells Ke Maa,b, Fengli Zhangb, Nima Sayyadi a, Wenjie Chena, Ayad G. Anwera, Andrew Carea, Bin Xub, Wenjing Tian*b, Ewa M. Goldys*a, Guozhen Liu*a,c a
ARC Centre of Excellence in Nanoscale Biophotonics (CNBP), Macquarie University, NSW 2109, Australia.
b
State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012, P. R. China. c
Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, P. R. China. KEYWORDS. IFN-γ detection, AIE fluorogens, aptasensor, bioimaging, turn-on fluorescence
ABSTRACT: We report an aggregation-induced emission fluorogen (AIEgen)-based turn-on fluorescent aptasensor being able to detect the ultra-small concentration of intracellular IFN-γ. The aptasensor consists of an IFN-γ aptamer labeled with a fluorogen with a typical aggregation-induced emission (AIE) characteristic, which shows strong red emission only in the presence of IFN-γ. The aptasensor is able to effectively monitor intracellular IFN-γ secretion with the lowest detection limit of 2 pg mL-1, and it is capable of localizing IFN-γ in live cells during secretion, with excellent cellular permeability and biocompatibility as well as low cytotoxicity. This probe is able to localize the intracellular IFN-γ at a low concentration less than 10 pg mL-1, and it is successfully used for real-time bioimaging. This simple and highly sensitive sensor may enable the exploration of cytokine pathways and their dynamic secretion process in cellular environment. It provides a universal sensing platform for monitoring a spectrum of molecules secreted by cells.
Cytokines are crucial cellular signaling molecules which are secreted by cells.1-3 They are present in body fluids and tissues at the pM concentration range,4-6 and elevated concentrations of cytokines normally accompany inflammation or disease.7 Interferon-gamma (IFN-γ), of specific interest in this study belongs to a group of cytokines with antiviral and antiparasitic activity and it also inhibits cellular proliferation.8-9 It is produced mainly by T-cells and natural killer cells activated by antigens, mitogens, or alloantigens.10-11 The normal method to analyze the expression of the IFN-γ is ELISA test, which is time consuming and not suitable for continuing monitoring. Cytokine monitoring is a significant but challenging area of biomedical sensing especially when in vivo and/or real-time sensing is required.12 Monitoring of the intracellular or extracellular IFN-γ is important to diagnose cancers, such as gastric and intestinal cancers.13-14 Due to their typically low levels (within pM range), one of the principal considerations is the detection sensitivity. Different strategies have been explored in this area to optimize sensitivity, such as immunosensing,15-16 electrochemiluminescence,17 DNAassisted methods and nanoparticles-based techniques.18 Aptasensors are well suited for sensing of small molecules due to their generally high affinity.19 In addition, aptamers are very stable in a biological environment compared to antibodies.20 Earlier published work from Pu’s group
demonstrated an electrochemical aptasensor for the detection of IFN-γ using graphene. The detection scheme was based on the exonuclease-catalyzed target recycling and TdT-mediated cascade signal amplification, and it achieved the lowest detection limit of 3 pg mL-1.21 Buntat et al. employed a prototype graphene based FET structure with a modified aptamer as biosensor for IFN-γ detection, with the sensitivity of about 10 μM.22 Revzin and coworkers designed several aptamer-based immunosensors to detect IFN-γ. They first reported an electrochemical DNA aptamer-based biosensor with a detection limit of 0.06 nM for IFN-γ detection in vitro.23 Subsequently, a microdevice was developed for the detection of local IFN-γ release from primary human leukocytes in real time, with the sensitivity of less than 60 pM.24 The same authors also used an electrochemical microsystem for simultaneously quantifying IFN-γ and IFN-α with the detection limits of 0.06 nM and 0.58 nM respectively.25 Chai’s group used a hairpin aptamer DNAzyme probe to detect IFN-γ with a sensitivity of 50 pM which could be detected by naked eye.26 Zhang’s group has reported an exonuclease III-aided cycle for amplified and label-free detection of IFN-γ with a theoretical detection limit of 0.1 pM and a visual detection limit of 20 pM by naked eye.27 However, none of these aptasensors are suitable for the monitoring of intracellular IFN-γ because of the requirement for enzymes, or large size of modified electrodes and dielectric layers. In addition, it is challenging to avoid
ACS Paragon Plus Environment
ACS Sensors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
the fluorescent quenching effect towards conventional fluorogens labelled on the aptamer. For aptasensors labeled with fluorophore and quencher, it is also difficult to achieve the absolute quenched fluorescence, which results in a large background signal lowering down the detection sensitivity. AIEgens are small fluorescent molecules sensors with a special switch-on emission property.28-29 The AIEgens comprise single bonds, which can freely rotate in the monomolecular state of an AIE-active sensor, preventing fluorescence emission when fully dissolved. However, the restriction of intramolecular rotations (RIR) occurs following aggregation, and thus, such aggregated AIEgens present strong fluorescence when poorly dissolved or in solid state.30-31 The AIE molecules offer a key advantage for biosensing and bioimaging due to their high brightness induced by aggregation and the relative immunity to fluorescence quenching.32-33 Besides the aggregation, any molecules attached to AIEgens which can restrict their rotation are also able to turn on their fluorescence.34-35 The AIEgens can be modified with different functional groups to enable the modification of biorecognition molecules to capture the analytes.36 These properties were utilized by Zhang’s group who modified an AIEgens with two peptides for ultrasensitive imaging of cancer cells.37 This AIE-based probe was able to recognize the LAPTM4B protein in the cancer cells. The attachment of this protein restricted the rotation of AIEgens and produced strong red emission. So far, to our knowledge, there are no reports of cytokine detection by using the AIEgens based biosensors. Herein, we developed an AIEgen-based fluorescent aptasensor (Figure 1) to detect trace levels of IFN-γ secreted by live cells. This aptasensor (referred to as TPE-aptamer) consists of an AIEgen named TPEN3 and an oligonucleotide which has high affinity to IFN-γ. TPEN3, is a tetraphenyl ethylene molecule modified with an azide group (Supporting Information, Figure S1). Due to good water solubility of the aptamer, this TPE-aptamer shows very low emission in the aqueous solution without IFN-γ. However, in the presence of IFN-γ, the TPE-aptamer interacts with IFN-γ through specific recognition between the aptamer and IFN-γ. The RIR happens due to the complexation between TPE-aptamer and IFN-γ, resulting in strong emission of TPE-aptamer. This aptasensor was applied here for in vivo detection of IFN-γ at the concentration as low as 10 pg mL-1. Other non-specific proteins did not show detectable interference to this aptasensor.
Figure 1. The schematics of the AIEgen-based fluorescent aptasensor for detecting intracellular IFN-γ.
The materials are detailed in the Supplementary Information. The synthesis process of compound 1, 2, 3, 4 and TPEN3 is shown in Figure S1 (Supporting information). The characteristics of these compounds are shown in Figure S2S7 (Supporting information). As shown in Figure S1, the aptamer was firstly conjugated with propiolic acid. To activate the carboxylic acid groups in the propiolic acid, 1 nmole propiolic acid, 4 nmole EDC and 2 nmole NHS were added to 400 μL water and mix-vibrated at room temperature for 20 min. Then 1 nmole aptamer (100 uL) was added into the mixture to react for another 2 h with shaking. The achieved aptamer modified with alkynyl group was used for further Click reaction to attach TPEN3. TPEN3 was diluted into 500 μL DMSO, and then alkynyl modified aptamer solution (500 μL) was added to maintain the H2O/DMSO ratio at 1:1. The 0.3 nmole copper sulfate and 0.6 nmole sodium L-ascorbate were added into the mixture to perform the Click reaction at room temperature for 24 h under shaking conditions. After that, the product was extracted with dichloromethane to eliminate the TPEN3 residue, and the remaining water phase was freeze-dried by lyophilizer to yield the final probe, referred to as the TPEaptamer. The final achieved TPE-aptamer is a mixture, including alkynyl modified aptamer, sodium L-ascortate and copper salt. While other substances in this mixture have no influence on bioimaging due to their rather low concentrations. To confirm the conjugation between the aptamers and TPEN3 molecules, we carried out electrophoresis of the aptamer and TPE-aptamer samples (Figure 2a). The TPEaptamers advanced somewhat more slowly than the control aptamers due to their slightly higher molecular weight, confirming the conjugation between TPEN3 and the aptamer. Moreover, some molecules remained on the origin for the TPE-aptamer band, indicating that some TPEaptamer molecules aggregated together. In comparison, no molecules remain for the aptamer band, because aptamer cannot aggregate. This aggregation phenomenon further confirmed the conjugation process. Figure 2b shows the absorption spectrum of the TPE-aptamer solution, with the peak centered at about 415 nm. Figure 2c shows the fluorescent emission spectrum of the TPE-aptamer in the mixture solution of H2O and THF (THF = 80%, v/v). The emission maximum was about 615 nm, representing a Stokes shift of about 200 nm.38
Figure 2. (a) Gel electrophoresis images for TPE-aptamer and aptamer; (b) The ultraviolet absorption spectrum of TPE-aptamer (1 nmole mL-1) in the water solution; (c) The fluorescent spectrum of TPE-aptamer (1 nmole mL-1) in the
ACS Paragon Plus Environment
Page 2 of 8
Page 3 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Sensors
solution containing H2O and THF (THF = 80%, v/v); Excitation: 415 nm. HPLC facility was also used to purify and confirm the achievement of TPE-aptamer. As shown in Figure S7a, the retention time of aptamer was 4.95 min at 260 nm major absorption (Supporting information). After the modification with propiolic acid, the retention time turned to 3.63 min at 260 nm major absorption, which indicated the modification of aptamer with alkynyl group (Figure S7b). The retention time of TPEN3 was 13.52 min both at 260 nm and 415 nm absorption (Figure S7c, d). The relative amount of absorption of TPEN3 at 260 nm was 28 mAU, but at 415 nm more than 250 mAU was observed, so the amount of UV absorption of TPEN3 at 260 nm was negligible compare to 415 nm. While the retention time of TPE-aptamer was 18.11 min both at 260 nm and 415 nm absorption (Figure S7e, f) due to the modification of TPEN3 with aptamers. The relative amount of absorption at 260 nm was around 350 mAU while it had around 700 mAU absorption at 415 nm, confirming the conjugation of aptamer with TPE. In order to establish the detection capability of the TPEaptamer towards IFN-γ, we used the 0.1 nmole mL-1 TPEaptamer solution to detect IFN-γ in the concentration range of 0-10000 pg mL-1. Figure 3a shows the fluorescence curves of TPE-aptamers after 1 h incubation with IFN-γ with different concentration at 37 oC. A gradual increase of fluorescence is observed with increasing concentration of IFN-γ because strong binding between the TPE-aptamer and IFN-γ induces the RIR, resulting in turning on of TPE fluorescence of the TPE-aptamer probe. As shown in Figure 3b, the emission intensity increases rapidly in the range from 0 to 1000 pg mL-1 of IFN-γ, and then the rate of increase slows down in the range from 1000 to 10000 pg mL1 until a plateau reached starting at the concentration of 2000 pg mL-1. This is because when the concentration of IFN-γ is above 2000 pg mL-1, a binding of TPE-aptamer with IFN-γ is saturated with the ignorable increase in the fluorescence intensity, and the intramolecular bonds rotation is further restricted by steric hindrance. It suggests that the fluorescence intensity of TPE-aptamer at 615 nm is independent of IFN-γ concentration once it passes the maximum binding ratio between TPE-aptamer and IFN-γ. A linear relationship between the fluorescence intensity and the concentration of IFN-γ from 0 to 100 pg mL-1 (R2 = 0.94853, Figure 3c) is observed. The error bars means three separate samples of TPE-aptamer mixed with three distinct IFN-γ samples. The limit of detection at signal-tonoise ratio (S/N) of 3 is estimated from three independent measurements to be about 2 pg mL-1. This indicates that the TPE-aptamer sensor is highly sensitive compared to other reported sensors listed in Table S1 (Supporting information).21-22 Only the presence of IFN-γ can trigger the strong emission of TPE-aptamer in the water solution by the binding between TPE-aptamer and IFN-γ and the subsequent happening of RIR effect. Other good solvent or other types of proteins in the water solvent have no effect towards IFN-γ detection because the fluorogen TPE-
aptamer is water-soluble and is has no emission in the water solution or other good solvent without IFN-γ. The purified TPE-aptamer doesn’t contain TPEN3 after the extraction process. Otherwise the fluorescent intensity in water might be high due to the AIE feature of TPEN3. The original intensity of TPE-aptamer is rather low, and increase accordingly after adding of IFN-γ (Figure 3). Other similar proteins do not induce the fluorescent enhancement suggesting the high specificity of TPE-aptamer towards IFN-γ (Figure 3d). IFN-γ was mainly produced by T-cells and natural killer cells, so PBMCs, a kind of T-cells were selected for bioimaging. However, these T-cells or natural killer cells were non-adherent cells which were flowing during real time imaging. Therefore, the adherent cells BV2 cells were used as the model in bioimaging for real time monitoring. And it was also possible to calculate the detecting sensitivity for the intracellular localization of IFN-γ by using BV2 cells treated with different concentration of IFN-γ. BV2 cells secrete negligible concentration of IFN-γ, and the BV2 cells were treated with IFN-γ protein before these experiments. To confirm that BV2 cells were able to take up IFN-γ, ELISA was used to detect the concentration of IFN-γ in the medium sample with or without BV2 cells after spiking in 300 pg mL-1 IFN-γ followed by incubation for 24 h. As shown in Figure S8 (Supporting information), the content of IFNγ remained at 294 pg mL-1 during the experiment in the absence of BV2 cells, while it decreased to 247 pg mL-1 in the presence of BV2 cells. This indicates that BV2 cells (8 × 105 cells mL-1) have uptaken about 47 pg mL-1 IFN-γ during the incubation. Subsequently, the previous culture medium was replaced by a fresh medium and the BV2 cells were incubated for another 24 h. After this second incubation, the concentration of IFN-γ in the medium was found to be about 35 pg mL-1, confirming that BV2 cells partly secreted out the uptaken IFN-γ into the medium, with some IFN-γ remaining inside BV2 cells (12 pg mL-1). We studied the stability of TPE-aptamer sensor in the culture medium. Figure S9 (Supporting information) shows the electrophoresis images of the aptamer in the culture medium or pure water. Both bands are well aligned, indicating that the aptamer was not hydrolyzed in the culture medium. Thus TPE-aptamer could be used directly to label the target in the cell culture medium.
ACS Paragon Plus Environment
ACS Sensors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Figure 3. (a) Graph of fluorescence spectra (excitation: 415 nm, emission: 615 nm) for TPE-aptamer probes (0.1 nmole mL-1) with the presence of different concentration of IFN-γ (0, 10, 30, 50, 70, 100, 300, 500, 700, 1000, 3000, 5000, 7000, 10000 pg mL-1) in the water solution; (b) The curve between fluorescence intensity and the concentration of IFN-γ obtained in (a); (c) The linear curve between fluorescence intensity and the concentration of IFN-γ from 0 to 100 pg mL-1; (d) The curve of the normalized fluorescence intensity (I-I0) for TPE-aptamer probes (0.1 nmole mL-1) with the presence of different proteins (IFN-γ, IFNβ, IFN-α, lgG or BSA; 100 pg mL-1); I0: fluorescence intensity in the absence of any protein. We then determined the ability of the TPE-aptamer sensors for in situ cytokine probing in BV2 cells. To this aim, the IFN-γ-treated BV2 cells were incubated with the sensors for different period of time (Supporting information, Figure S10). The turn-on fluorescence was observed after incubation the cells with the TPE-aptamer for 2-8 h. Thus 2 h was chosen as preferred incubation time for bioimaging. For the imaging experiment, different concentration of IFN-γ was added to the BV2 cell culture medium and incubated for 24 h. After that, 0.1 nmole mL-1 of the TPEaptamer was added to the BV2 cells for 2 h at 37 oC followed by carefully washing with PBS. As shown in Figure 4, a significant TPE-aptamer fluorescence was observed in BV2 cells. Two concentrations of IFN-γ (10 or 20 pg mL-1) were used to treat BV2 cells. According to the data in Figure S8 (Supporting information), BV2 cells only took up 47 pg mL-1 after treated with 300 pg mL-1 IFN-γ. Thus not all IFN-γ was taken up by BV2 cells which suggested that the absorbed IFN-γ was much less than 10 or 20 pg mL-1 (Figure 4). With the increasing in the concentration of IFN-γ from 10 pg mL-1 to 20 pg mL-1, the fluorescence intensity (Figure 4a) and labeling efficiency (Figure 4b) calculated with “Image J” software increased significantly, indicating the successful intracellular IFN-γ probing in live cells by TPE-aptamer. The labeling efficiency was about 10% when 20 pg mL-1 IFN-γ was incubated with BV2 cells, which contributed to the rather low level of IFN-γ uptaken by BV2 cells, resulting in a lower labeling efficiency compared to
that of PBMC cells. It was clear that the red fluorogens aggregated in the cytoplasm, not in the cell nucleus. Therefore, the cell-absorbed IFN-γ existed in the cytoplasm and could be recognized by TPE-aptamer. TPE-aptamer first accessed the cytomembrane through endocytosis, and then localized by IFN-γ to achieve red emission.39 As controls we used BV2 cells where another cytokine IFN-α which has no affinity to our aptamer was added to the BV2 cells (Supporting information, Figure S11). We found that only IFN-γ-treated, and not IFN-α-treated BV2 cells were labelled. Another control study was carried out by staining the IFN-γ treated BV2 cells with the TPE-aptamer or TPEN3 (Supporting information, Figure S12). No fluorescence labeling was observed with the treated BV2 cells in the presence of TPEN3. Additionally, the uptake efficiency of TPE-aptamer was measured by ultraviolet absorbance analysis. The absorbance of TPE-aptamer probe (0.1 nmole mL-1) in the PBS buffer was around 0.02202, and it decreased to 0.00154 after the staining of BV2 cells for 2 h (Supporting information, Figure S13). The uptake efficiency of TPE-aptamer was thus estimated to be 93%. These results demonstrated that the TPE-aptamer sensor was efficiently transported through the cell membrane and able to detect IFN-γ intracellularly.
Figure 4. Confocal laser scanning microscope images (ab, c-d, e-f) of BV2 cells treated with different concentration of IFN-γ (0, 10, 20 pg mL-1) for 24 h, then stained with the TPE-aptamer probes (0.1 nmole mL-1) for 2 h; Blue emission is from Hoechst 33342 (5 µg mL-1) with 430-470 nm emission range; Scale bars: 20 μm, PBS buffer; (g) Mean fluorescence intensity and (h) labeling efficiency of the TPEaptamer in BV2 cells with the increasing concentration of IFN-γ (0, 10, 20 pg mL-1); *p < 0.05, **p < 0.01 (t test) compared with the BV2 cells with 0 pg mL-1 IFN-γ.
ACS Paragon Plus Environment
Page 4 of 8
Page 5 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Sensors
Figure 5. Confocal laser scanning microscope images of PBMCs after stained with 0.1 nmole mL-1 TPE-aptamer (ab, c-d) or 0.1 nmole mL-1 TPEN3 (e-f) for 2 h; Blue emission is from Hoechst 33342 (5 µg mL-1) with 430-470 nm emission range; Scale bars: (a, c, e) 20 μm, (b, d, f) 10 μm, PBS buffer. PBMCs were able to secrete IFN-γ without stimulation (14.15±13.77 pg mL-1 at cell density of 5 × 105 cells mL-1),40 and also after LPS stimulation. Our results confirmed that, after incubation of unstimulated PBMCs (8 × 105 cells mL1) with TPE-aptamer for increasing periods of time, the levels of IFN-γ secreted from PBMCs increased (Supporting information, Figure S14). The concentration of IFN-γ was about 57 pg mL-1 after 24 h incubation, which was within the sensitivity range of the prepared TPE-aptamer sensors. Due to the higher concentration of IFN-γ secreted from the cells, the labeling efficiency of PBMCs with the TPEaptamer was also higher (about 70%, Figure 5) than in IFNγ-treated BV2 cells. As controls, the TPE-aptamer and the TPEN3 (AIE molecules but without the attached aptamers) were respectively used to label PBMCs and we found that only the TPE-aptamer could successfully label the PBMCs (Figure 5). A kind of cancer cells named Raji cells were also be used as control cell lines for studying the performance of TPE-aptamer for probing IFN-γ. As shown in Figure S15 (Supporting information), there were none labeled cells because these cells contained few IFN-γ.41 This confirmed that the TPE-aptamer was capable to specifically recognize IFN-γ. Confocal microscopy imaging of BV2 cells was carried out over the course of the labelling process. The BV2 cells were first treated with IFN-γ (20 pg mL-1) for 24 h at 37 oC, followed by the addition of the TPE-aptamer (0.1 nmole mL-1) at 37 oC and incubation for varying periods (0-120 min). The cells were expected to be labeled with TPEaptamer if intracellular IFN-γ was present, and no fluorescence was expected if the cytokine molecules were outside the cells. As shown in Figure 6a, no cell labeling was observed after incubation the BV2 cells with the TPE-aptamer sensors for 0-30 min. However, a weak signal appeared after incubation for 45 min, which could be observed in confocal microscopy. This observation suggested that it took more than 30 min for the sensors to penetrate the cells and
Figure 6. (a) Time course confocal imagings of BV2 cells stained with TPE-aptamer (0.1 nmole mL-1) in the presence of IFN-γ (20 pg mL-1); Scale bars: 10 μm, DEME medium; Arrows showing the turn-on fluorescence of TPE-aptamer probes (b) Mean fluorescent intensity of TPE-aptamer in BV2 cells with the increasing incubation time; Times: 0, 15, 30, 45, 60, 75, 90, 105, 120 min; **p
