Multiplexed Analysis for Anti-Epidermal Growth Factor Receptor

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Multiplexed Analysis for Anti-EGFR Tumor Cell Growth Inhibition Based on QD Probes Dahai Ren, Yiqiu Xia, Bin Wang, and Zheng You Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04471 • Publication Date (Web): 28 Mar 2016 Downloaded from http://pubs.acs.org on April 1, 2016

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Analytical Chemistry

Multiplexed Analysis for Anti-EGFR Tumor Cell Growth Inhibition Based on QD Probes Dahai Ren*, Yiqiu Xia, Bin Wang, Zheng You State Key Laboratory of Precision Measurement Technology and Instruments, Department of Precision Instrument, Tsinghua University, Beijing, 100084, China

Abstract: Quantum dot (QD) multi-color analysis was achieved by labeling the epidermal growth factor and labeling the antibody against the epidermal growth factor receptor (anti-EGFR) using different QDs. Based on the fluorescence intensity of the two types of QDs, the amount of EGFs in the cells and anti-EGFRs bound to EGFRs were analyzed. The functions of anti-EGFR in preventing HeLa cells from engulfing EGF and inhibiting over-proliferation of the HeLa cells were also studied. Meanwhile, parallel analysis was conducted to analyze the heterogeneity of cells at the single-cell level using a single-cell array. This provides a novel approach for the multiplexed analysis for the anti-EGFR functions in cells together with the cell heterogeneity. It also lays a foundation for parallel analysis and detection using various fluorescence probes, simultaneous tracking and detection of multiple targets at an overall level or individual level, and multi-channel analysis of drug effects.

E

pidermal growth factor receptor (EGFR) is an expression product of proto-oncogene c-erbB1 and is widely distributed on the surface of epithelial cells, horny cells, glial cells and fibroblasts, etc. The

relative ligand epidermal growth factor (EGF) can bind EGFR to form a dimer, which will be engulfed by cells after autophosphorylation1. It can activate the downstream signal pathway, resulting in higher calcium level in the cells2,3. The glycolysis and protein synthesis is enhanced and the gene expression is improved, prompting cells into cell cycle progress, which results in cell reparation, differentiation, proliferation, migration, adhesion and attack (Figure 1). In some cancer cells, EGFR is always over-expressed4-6. For instance, the percentage of EGFR expression is 36-100% in head and neck neoplasms, 90% in cervical cancer, 40-80% in prostate cancer and lung cancer, 33-74% in stomach cancer, 47-68% in liver cancer, 50-90% in renal carcinoma, 43-89% in esophageal cancer, 35-70% in ovarian cancer, and 35-86% in bladder cancer. The over-activation of these receptors inhibits cancer cell apoptosis and promotes cancer cell proliferation, and also promotes cancer cell migration, adhesion and hemangioma formation7. There is an important category of medicine for these cancers that targets EGFR, preventing cancer cells from over-growth and proliferation by inhibiting the expression of EGFR, the binding of ligands and EGFR or the transmission of downstream signals8-10. The cytotoxicity and clinical toxicity of anti-EGFR drugs have been studied in the traditional way11,12. When peripheral blood mononuclear cells (PBMC) were cultured with anti-EGFR drugs for 72h, no enhancement of PBMC cytotoxicity occurred and anti-tumor PBMC activity was reinforced11. At present,

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including

anti-EGFR,

is

MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole) assay, which is used to measure the activity of cellular enzymes that decrease the tetrazolium dye by microplatereaders13,14. However, in MTT assays, cells are always damaged. In addition, we can only obtain the overall information of the cells. In this paper, we adopted QD probe for analysis, which is less harmful compared with MTT assay. Moreover, a long observation period is enabled and multiple labeling and analysis on single cell level can be achieved.

Figure 1. Schematic diagram of EGF Effects.

QD is a new fluorescent material. Due to their quantum scale effect and wide excitation wavelength range, various QDs can be excited with the same light source15-17 to observe multiple targets at the same time without special requirements for excitation light sources or filters18-20. Additionally, QD features high fluorescence intensity (its single molecule fluorescence intensity is 20 times that of Rhodamine 6G)21,22 and high photobleaching resistance (its photobleaching resistance time is 100 times that of Rhodamine 6G)23,24, allowing long time tracking and observation for one material25,26. This makes QDs an excellent fluorescence labeling material for dynamic fluorescence imaging, target tracing and material testing in biomedical research, and provides a new opportunity for the development of biochips and biosensors27-30. In this paper, QD probes emitting two types of fluorescence were used to carry out multi-color labeling of the receptor and ligand closely associated with cell growth and proliferation. The effects of drugs on cells were analyzed via the fluorescence intensity of the probes. The paper discusses an approach for conducting multi-channel parallel analysis on cell behaviors and drug functioning using these probes. Specifically, the study is based on the principle that the anti-EGFR inhibits the binding between the EGF and the EGFR. QDs emitting different fluorescence were used to study the effects of the anti-EGFR.


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In this paper, drug effects were evaluated using various QD probes. The fluorescence intensity was measured to evaluate the effects. The parallel analysis functions of QDs emitting different fluorescence were utilized. A QD-based multi-channel parallel analysis was also conducted to analyze the cell behaviors and drug effects at the single-cell level. Furthermore, single-cell arrays were used for the analysis, making the simultaneous analysis for the cell heterogeneity possible. This provides a novel approach to analyze the anti-EGFR functions in cells together with the cell heterogeneity. QDs emitting more fluorescence colors can be used in the same way to analyze multiple targets so as to improve testing efficiency.

EXPERIMENTAL SECTION Materials, cells and tools. Streptavidin, Streptavidin-CdS QD625, phosphate buffer solution (PBS) (PH=7.2~7.4), Epidermal Groeth Favtor biotin-XX conjugatue (biotin EGF), EGFR AB finity Rec RB Mono AB EA (the primary antibody), Donkey anti-Rabbit IgG (the secondary antibody). The HeLa cell was maintained in a DEME medium with 15% (v/v) Fetal Bovine Serum (FBS) and 1% (v/v) Penicillin-Streptomycin Solution. The cells were cultured in a humidified atmosphere at 37OC with 5% CO2. The cells were observed with an inverted fluorescence microscope (Olympus IX71). The BP470-495 excitation filter and the BA510-IF emission filters were used along with software for capturing images with the microscope. Preparation of QD probes. We first synthesized CdSe QDs in ODE and TOPO systems31. To enlarge the fluorescent range and get more fluorescent colors, we utilized paraffin liquid as the solvent instead of QDE and oleic acid (OA) as the stabilizer instead of TOPO. Finally, we obtained CdSe QDs emitting colors ranging from purple to red in a simple and green experimental condition, to reach the requirements of multiplexed detection. By utilizing this method, we synthesized high-quality QD probes for multi-analysis that are not only low cost, but less contaminating and dependent on equipment. Cytotoxicity analysis of the probes. To evaluate the toxicity of the quantum dots made in this study, three complementary approaches were applied, including cell morphology, trypan blue staining assay, and MTT assay. Hela cells were divided into an experimental group and a control group, both of which were cultured in 96-well plates. Appropriate amount of biotin-EGF and 0.5pmol QDs conjugated with streptavidin were added to the experimental group, where only biotin-EGF with the same concentration was added into the control group. Both groups were cultured for 24h in an incubator. EGFR Labeling on the Cell Surface. QD-EGFR was first labeled by the secondary antibody, and then labeled by the primary antibody. Here, we incubated the primary antibodies and QD-EGFR conjugated with secondary antibodies together in PBS medium for 1 hour (at a ratio of 1:1) and then the secondary antibodies were bound to the primary antibodies. Moreover, when the secondary antibody was added to the solution of QD-EGFR labeled by the primary antibody, the catalyst EDC had already been hydrolyzed and lost its activity. Therefore, the free secondary antibody could not be conjugated to the surface of QDs, ensuring the specificity of the probes. Then, we added the incubation medium to the cell medium, and



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incubated for 1 hour. After incubation, the compounded primary and secondary antibodies were used to label the EGFR on the cell surface. The cells were observed under a microscope after they were cleaned using PBS (Figure 7B). The figure shows an intense green fluorescence on the cell surface, while no big green fluorescence zone was found at other locations outside the cells. This indicates that the EGFRs on the cells surfaces have been successfully labeled by the secondary antibodies linked with green QDs. (Detailed process, experiment results and figures can be found in Supporting Information.) Cells patterning. To analyze the heterogeneity of cells, they were separated and located by cell patterning technology. We developed a convenient single-cell patterning method. A series of micro-wells were fabricated for single-cell patterning. Under microfluidic conditions, cells were introduced to the surface of this micro-well array. Because of the gravitational force, cells were confined to the micro-wells and the unfixed cells were washed away. This approach is simpler than the biotin-(strept)avidin system we have employed32,33.

RESULTS AND DISCUSSION Impacts of EGF and Anti-EGF on Cells. We investigated the impacts of EGF on Hela cells and the influence of EGF concentration. Experiments were carried out with 4 different EGF concentrations during 29 hours, indicating that the cells grew very slowly and rarely adhered to the wall if no EGF was present. However, with the increase of the EGF concentration, the cells grew faster and more cells properly adhered to the wall. The cell growth was also observed for the four groups after 43 hours. Experiments indicated that, the EGF can not only fasten the cell cycle process, promote cell growth, adherence and adhesion, but also effectively prevent cell apoptosis. Additionally, the results of the four groups also make it easier to determine the desired EGF concentrations for drugs. (Relevant experiment results and figures can be found in Supporting Information.) We investigated the impacts of anti-EGF on Hela cells. Experiment indicated that with the increase of anti-EGFR concentration, the cell growth and proliferation rate decreases. When 1.50µL (0.5µg/µL) anti-EGFR is added, the cell growth rate is only 63% of normal value. Here the cell growth rate was referred to MTT assay absorbance ratio of the experimental group and negative group, which could characterize the cell proliferation rate. This is also consistent with the previous analysis results of anti-EGFR concentrations and the amount of engulfed EGF calculated by the fluorescence intensity. In this manner, we can identify anti-EGFR drugs’ inhibition of cell growth and proliferation by above fluorescence detection methods (Figure S1-4 in Supporting Information). Cytotoxicity of QDs to HeLa cells. First, we determined the cytotoxicity of QDs by investigating the morphology of treated cells. Both control and QD-treated cells exhibited normal adhesion and cell morphology, indicating that QDs were not toxic to the cells (Figure 2A).


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Figure 2. Cytotoxicity of QDs to HeLa cells. (A) Hela cells’ outlines after 24h of incubation. (A1) Absence of QDs; (A2) with 5nM QDs, bar = 100µm. (B) Survival rate of Hela cells with 5nM QDs and without QDs. (C) Absorption values of Hela cells after cultured with 5nM QDs (cell free samples with 5nM QDs were used as controls). The paired t-test between experimental group and control group was calculated and p value was 0.012, which confirmed that QDs truly have inhibitory effects on cell growth and proliferation to some extent. However, as shown in the scatter plots, it was obvious that the inhibitory effect was very weak.

Second, we evaluated the viability of HeLa cells by staining the cells with typan blue (Equation 1). The results showed that 100% and 97.7% cells remained viable after being cultured without or with QDs, respectively (Figure 2B), which further evidenced that QDs were not toxic to the cells.

 number of cells dyed by trypan blue  cell ' s living rate = 1 −  ×100% total number of cells  

(1)

Furthermore, we investigated if QDs have any inhibitory effects on cell growth and proliferation using MTT cell activity assay (Equation 2). After culturing for 24h, both the experimental group and control group were added with 10µL MTT and incubated in dark at 37°C for 4h, by which MTT was reduced to Formazan. Then 100µL dimethyl sulfoxide was added and the samples were incubated in dark at 37OC for 8h to fully dissolve Formazan. A microplate reader was used to record the absorption at the wavelength of 570nm, and the results were compared with the value at the wavelength of 630nm. To eliminate the background signal of QDs, 5nM QDs were also added to the control group. The results show that the growth rate was repressed by



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5.8% in the presence of QDs (Figure 2C). Original data of experimental and control groups are presented in Figure S7 in Supporting Information.

Cell ' s inhibition rate  absorbance value of experimental group − absorbance value of blank group  = 1 −  absorbance value of control group − absorbance value of blank group   ×100%

(2)

Detection of EGF’s Effect with QDs. Cell growth can reflect the impacts of EGF-inhibiting drugs. However, if fluorescence is used to label the EGF, the fluorescence intensity could visually reflect the impact of the EGF on the cells, making it easier to observe the impact. Biotin EGF (a type of biotinylated EGF) was labeled using streptavidin-QD625 (red fluorescence) to obtain QD625-EGF emitting red fluorescence. We studied the ratio of biotin streptavidin-QD625/ EGF to determine the desired value, making it easier to observe the utilization of EGF by the cells. We added 12.5nM biotin EGF, 1.25nM, 2.5nM and 5.0nM streptavidin-QD625 to the three groups of 100µL cell suspension. The cells were cleaned by PBS after being cultured for 24 hours to remove the labeled QDs and then observed under a fluorescence microscope (Figure 3A). As shown in the fluorescence images for the three groups, no fluorescence was found when the ratio of streptavidin-QD625/ biotin EGF was 1:10. When the ratio was 1:5 and 1:2.5 respectively, a red fluorescence was found in the cells, indicating that QDs had entered the cells along with the EGF. When the ratio was 1:5, intense fluorescence was found. As a result, the ratio 1:5 could be used as a reference value for further experiments. The utilization of EGF by the cells was observed after they had been cultured for a night, with the forementioned streptravidin-QD625/ biotin EGF ratio of 1:5. The control group (Figure 3 B1 & B2) contains only streptavidin-QD625 of the same concentration and was biotin EGF-free. The experimental group (Figure 3 B3 & B4) contains 12.5nM EGF. As shown in the final fluorescence images (Figure 3B), no fluorescence was found in the cells in the control group, while an intense red fluorescence was found in the cells in the experimental group, and the fluorescence was within the cell contour, especially around the center. This corresponds with the phenomenon that the EGF would enter the nucleus if it enters the cell. We can rule out the possibility that the QDs were non-specifically bound or engulfed by the cells through comparison between negative control and experimental control, as QDs could not attach to or get into the cell without conjugating to biotin-EGF. This means that QDs can be used to label the EGF, and the amount of EGF utilized by the cells can be reflected by the fluorescence intensity of QDs.


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Figure 3. Detection of EGF’s effect with QDs. (A) Labeling of the EGF impacts on the cells at different QD concentrations (EGF concentration: 12.5nM) Magnification: 400x, Scale: 100µm; Exposure time: 500ms. (B) Observation of EGF impacts on the cells labeled by QDs. B1& B2: EGF-free; B3 & B4: 12.5nM EGF; Magnification: 400×; Scale: 100µm; Exposure time: 500ms.

Impacts of Drugs on the binding of EGF and EGFR. After being bound to the EGFR, the EGF is engulfed by the cells, initiating signal transduction. A large amount of EGFRs were present on the cell membrane surface, and many EGFs were engulfed due to abnormal expression of the EGFR on cancer cells. This results in massive cell growth, migration, and apoptosis inhibition. EGFR-inhibiting drugs are a crucial type of anti-cancer drugs, designed to cure abnormal EGFR expression. The principle of EGFR-inhibiting drugs is as follows: An EGFR-inhibiting agent, such as anti-EGFR, binds with the EGFR



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to prevent cells from engulfing the EGF after it binds with EGFR. This breaks signal transduction, prevents the cells from entering the growth cycle, and promotes cell apoptosis (Figure 1). Red fluorescence QD625 was used to label EGF, for which different concentrations of anti-EGFR were applied. The purpose is to study the impacts of anti-EGFR on the growth of cancer cells, i.e. the inhibition against the growth cycle of these cells. The amount of EGFs in the cells was judged by the fluorescence intensity. In addition, we labeled anti-EGFR with green fluorescence QD525 to learn the binding between the EGFR and anti-EGFR of different concentrations (Figure 4). The purpose is to learn the changes in the amount of EGF in the cells that caused by the impact of anti-EGFR. Information about binding was obtained based on the intensity of the green fluorescence on the cell membrane.

Figure 4. Principle for testing anti-EGFR effects.

HeLa cells were cleaned with PBS three times to remove the original culture medium. Then, a serum-free culture medium was added to ensure that the environment where cells grew was free of EGF (cell concentration was1×104/ml). After that, the medium was divided into six groups, which contained 1.25ng/µL, 2.50ng/µL, 3.75ng/µL, 5.00ng/µL, 6.25ng/µL, 7.50ng/µL anti-EGFR Rabbit IgG respectively (Note: The anti-EGFR Rabbit IgG was bound with QD525 Donkey anti-Rabbit IgG. The concentration of anti-EGFR was 0.5µg/µL, and cell solution volume was 100µL). The QD525 Donkey anti-Rabbit IgG was incubated with the cells for 1 hour so that the anti-EGFR contacted the cells sufficiently. A QD625-EGF was then added (EGF content was 12.5nM). The cells were cleaned by PBS after culturing for a night. The cells were then observed under a microscope (Figure 5A).


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Figure 5. Fluorescence of cells when different concentrations of anti-EGFR were added. (A)Fluorescence images of cells when different concentrations of anti-EGFR were added. A: 1.25ng/µL; B: 2.5ng/µL; C: 3.75ng/µL; D: 5.00ng/µL; E: 6.25ng/µL; F: 7.50ng/µL. EGF content: 12.5nM, Magnification: 400x, Scale: 100µm, Exposure time: 500ms. (B) Anti-EGFR contents versus anti-EGFR-QD fluorescence intensity on cell surface. (C) Anti-EGFR contents versus QD-EGF fluorescence intensity in cells.



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As seen in Figure 5B, the intensity of the green fluorescence around the cell increased, as the amount of anti-EGFR increased (from A to F). The increase in the fluorescence intensity was obviously caused by more bindings of anti-EGFR and EGFR. In contrast, the intensity of red fluorescence in the cells decreased gradually as the amount of anti-EGFR increased, which suggested a decreased amount of EGF in the cells (Figure 5C). Scatter plots of Figure 5B and Figure 5C are shown in Figure S8 in Supporting Information. We can conclude that less EGFs were engulfed by the cells, and the chances of binding between EGF and EGFR decreased because more EGFRs on the cell surface were occupied by the anti-EGFR as the amount increases. To reflect the amount of the protein labeled by probes with the fluorescence intensity of QDs, the intensity of the red fluorescence in the cells cultured with 6 anti-EGFR concentrations was calculated (Equation 3) and the intensity of the green fluorescence around the cells (Equation 4) with software ImageJ34. Corrected red fluorescence inside the cell = sum of the intensity of the red pixels inside the cell − area of red fluorescence ×

∑ sum of the intensity of the pixels per unit area around the region of red fluorescence

(3)

3

Corrected green fluorescence intensity around the cell = sum of the intensity of the pixels for one cell

∑ sum of the intensity of the pixels per unit area around the cell

− area of the cell × ∑

3

(4)

− Corrected red fluorescence inside the cell

As shown in Figure 5B, with the increase in anti-EGFR concentration, the intensity of the green fluorescence around the cells increases continuously. But, the increment is reduced gradually when the anti-EGFR concentration is higher, indicating that the EGFRs on cell surfaces are getting saturated. Additionally, as shown in the figure, the increment of the intensity of the green fluorescence around the cells is less than that of anti-EGFR, which could be intuitively explained using Langmuir isotherm in chemical kinetics. As shown in Figure 5C, with increase in anti-EGFR concentration, the intensity of the red fluorescence in cells continuously decreases. This means the amount of the EGFs engulfed by cells also decreases, and the decrement is reduced gradually when the anti-EGFR concentration is higher. With a high anti-EGFR concentration (higher than 1.25µL), there are less empty EGFRs on the cell surface. Less EGFs can be engulfed, and additional anti-EGFRs have less influence on EGF engulfment by the cells. All these results are consistent with the analysis results of Figure 5B. However, the initial decrement in the intensity of red fluorescence in the cells was greater than the increment in the anti-EGFR’s green fluorescence intensity. This may be related to the fact that EGFRs recycled to the cell membrane to bind new EGFs, instead of degradation after binding with EGF and endocytosis35. In the analysis above, we obtained the amount of the EGFR-bound anti-EGFRs on the cell surface and the amount of the EGFs engulfed by cells by using the fluorescence intensity of green and red QDs. The amount of probe targets can be obtained by measuring the fluorescence intensities of various QDs for analysis.


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Anti-EGFR Impact Analysis on the Single-Cell Level Based on Conventional Chips. Different from the overall analysis of cells, for individual cells, the amount of EGFRs expressed on the surface of different cells typically differs, or even differs significantly, due to the heterogeneity of cancer cells. Heterogeneity among cancer cells arises as a consequence of genetic change, environmental differences and reversible changes in cell properties36. Therefore, even with the same concentration of anti-EGFR, the intensity of the red fluorescence in cells may differ, and even the intensity of the green fluorescence on the cell surface also differs significantly (Figure 6A), which indicates the differences among cells in the amount of EGFR on the cell surface and EGF engulfing capability. We selected 20 cells, and measured the intensity of the green fluorescence on the cell surface and the red fluorescence in cells with the above method to obtain the relative amount of the EGFR bound anti-EGFR on the cell surface and the relative amount of the EGFs engulfed by cells (Figure 6B). We also calculated the ratio of green to red fluorescence intensity in these cells (Figure S5) and drew the boxplot to further analyze the fluorescence intensity distribution (Figure S9A). As shown in the figure, for most cells, the amount of EGFR-bound green fluorescence anti-EGFRs on the cell surface is the same, with a fluorescence intensity of approx. 26,000 (a.u.). The value is consistent with the intensity value obtained with the same concentration of anti-EGFR. Additionally, for these cells with the same green fluorescence intensity, the amount of red fluorescence EGFs in most cells is also relatively consistent, with a fluorescence intensity of approx. 17,000 (a.u.). This suggests that despite the great heterogeneity of cancer cells, there is no big difference among most cells in this regard. Additionally, this also verifies the reliability of the above method, which deducts the overall behaviors of cells based on randomly selected cells. However, for cells with the same anti-EGFR fluorescence intensity of approx. 26,000 (a.u.), some have a lower EGF fluorescence intensity. This may be caused by slow engulfment of EGF by the cells, whose surface only has a small amount of EGFRs, under the influence of anti-EGFR. Similarly, for the cells with low anti-EGFR fluorescence intensity, some have a lower EGF fluorescence intensity. This is also caused by slow EGF engulfment, as only a small amount of EGFRs is present on the cell surface. In addition, the anti-EGFR and EGF fluorescence difference between these cancer cells is also related to the amount of the EGFRs on the cell surface, EGFR cycling to the cell surface after endocytosis, and the binding of EGFR and EGF.



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Figure 6. Anti-EGFR impact analysis on the single-cell level. (A) QD625-EGF Utilization by the cells under impacts of QD525-antiEGFR (anti-EGFR: 0.25µL; EGF: 12.5nM) Magnification 640×; Scale: 62.50µm; Exposure time: 750ms. (B) Anti-EGFR fluorescence intensity versus EGF fluorescence intensity on a single cell level.


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Anti-EGFR Impact Analysis Based on a Single-Cell Array. Micropatterning techniques provides more accurate tools in biological mechanism, drug effect and biochemical reaction research. Micropatterning methods can enhance the sensitivity, the efficiency and the integration scale of biosensors. Moreover, by micropatterning techniques, the heterogeneity of cells can be further analyzed, which is very difficult in most cases37-39. A micro-holes array was fabricated with PDMS, and the cells medium was injected with a micro flow channel, by which the single-cell array was obtained (Figure 7A). We analyzed anti-EGFR’s inhibition of the growth and proliferation of HeLa cells using a single-cell array. Similarly, we labeled EGF with red fluorescence QD625, and labeled anti-EGFR with green fluorescence QD525. Anti-EGFR’s inhibition of EGF engulfment by the cells was identified at a single-cell level using the method for fluorescence intensity calculation stated previously (Figure 7B). As shown in the figure, an intense red fluorescence was found on all cells, while a weak green fluorescence was present on these cells. The intense red fluorescence was caused by massive engulfment of EGFs by HeLa cells due to the weak inhibition of the anti-EGFR as only a few EGFRs were occupied on the cell surface under the initial concentration of anti-EGFR (0.25µL, 0.5µg/µL). For individual cells however, the intensities of green fluorescence on the cell surface and red fluorescence in the cell are different (Figure 7D). This clearly indicates the differences among cells in the amount of EGFR on the cell surface and EGF engulfing capability. Therefore, the effects of anti-EGFR drugs on the individual cells are different. These conclusions are consistent with the above analysis of the drug impacts on HeLa cells. Therefore, individual behaviors and heterogeneity of cells at a single-cell level can be properly analyzed on a single-cell array. The single-cell array surface was not pretreated, which was unfavorable for the growth of HeLa cells given their poor adherence ability. This indicated that the cells were not in normal growing conditions. As a result, the drug effect evaluation for HeLa cells as shown in Figure 7B was affected. Additionally, during the evaluation, the cells must grow normally so that the growth and proliferation abilities can be further identified. To improve the effects of single-cell array analysis, the base surface should be treated to approximate the cell growing environment and culture cells on the single-cell level. In addition, the treatment also allows the tracking and observation of cell behaviors and drug effects during growing and proliferation.



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Figure 7. Anti-EGFR’s impacts on EGF engulfment by HeLa cells using single-cell array analysis (anti-EGFR: 0.25µL (0.5µg/µL); EGF:12.5nM). (A) Single-cell array. Magnification 400×, Scale: 100µm. (B) Fluorescence image. Magnification 400×; Scale:100µm; Exposure time: 100ms. (C) Bright field image. Magnification 400×; Scale: 100µm; Exposure time: 100ms. (D) Anti-EGFR fluorescence intensity versus EGF fluorescence intensity on a single cell level, indicating tumor cells heterogeneities. The boxplot of green and red fluorescence intensity was showed in Figure S9B in Supporting Information.


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CONCLUSIONS In this paper, we labeled the EGF with red fluorescence QD625 to obtain probes QD625-EGF, and observed EGF engulfing during HeLa cell growth progress to learn the utilization of the EGF by the cells. Simultaneously, we labeled anti-EGFR with green fluorescence QD525 to obtain probes QD525-antiEGFR, and learnt the binding of anti-EGFR and the EGFR. The purpose is to learn the changes in the amount of EGF in the cells that caused by the impact of anti-EGFR, which has not been effectively studied before. Furthermore, using the two bio-probes simultaneously, the effects of anti-EGFR drugs were analyzed by measuring changes in the intensities of red and green fluorescence when different amount of anti-EGFR drugs was added at a single-cell level. At the same time, the drug effect was further analyzed at single-cell level using a single-cell array, and the heterogeneity of the cells was analyzed. The experimental results show that the number of targets can be obtained by measuring the fluorescence intensities of different QD probes. This allows multi-channel analysis and parallel analysis. Compared with the most common method - MTT assay, this method is less harmful and the real-time observation can be achieved in over a long period. Moreover, we can also multiply label proteins on single cells, allowing multiplexed analysis on a single cell level, which is of great significance to the drug screening.

ASSOCIATED CONTENT Supporting Information Additional information including the impacts of EGF and Anti-EGF on Hela cells, EGFR labeling on the cell surface and statistics Analyses of QD cytotoxicity and tumor cell heterogeneity. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work is supported by National Natural Science Foundation of China (No. 61071002), National Program for Significant Scientific Instruments Development of China (No. 2011YQ030134), the Funds of State Key Laboratory of China, the Scientific Research Foundation for Returned Overseas Chinese Scholars and the Funds for Beijing Laboratory of Biomedical Detection Technology and Instrument. We also thank Professor Yinye Wang of Beijing University for the cell supports.



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