Article pubs.acs.org/ac
Pattern Recognition of Cells via Multiplexed Imaging with Monosaccharide-Imprinted Quantum Dots Shuangshou Wang, Yanrong Wen, Yijia Wang, Yanyan Ma, and Zhen Liu* State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China S Supporting Information *
ABSTRACT: Recognition of cancer cells is essential for many important areas such as targeted cancer therapy. Multimonosaccharide-based recognition could be a useful strategy to improve the recognition specificity, but such a possibility has not been explored yet. Herein we report pattern recognition of cells via multiplexed imaging with monosaccharide-imprinted quantum dots (QDs). Imprinted with sialic acid, fucose, and mannose as the template, respectively, the QDs exhibited good specificity toward the template monosaccharides. Multiplexed imaging of cells simultaneously stained with these monosaccharide-imprinted QDs revealed the relative expression levels of the monosaccharides on the cells. Pattern recognition constructed using the intensities of multiplexed imaging unveiled the similarities and differences of different cell lines, allowing for the recognition of not only cancer cells from normal cells but also cancer cells of different cell lines. Thus, this study paved a solid ground for the design and preparation of novel cancer-cell targeting reagents and nanoprobes.
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specific to monosaccharide have been developed.28,29 Particularly, monosaccharide-specific MIPs have been facilely fabricated via a general approach called boronate affinity oriented surface imprinting30−32 and employed for the recognition and imaging of cancer cells over normal cells.33,34 The monosaccharide-specific MIPs exhibited better specificity than lectins, which are widely used for carbohydrate recognition. Single SA-, Fuc-, or Man-imprinted MIPs were able to differentiate cancer cells from healthy cells. Since antibodies against carbohydrates are hard to prepare,35 monosaccharide-specific MIPs hold great promise for cell recognition, and it is worthy to explore the feasibility of multimonosaccharide-based cell recognition strategy. In this study, we present pattern recognition of cells via multiplexed imaging with three types of monosaccharideimprinted quantum dots (QDs). QDs were employed as a scaffold for the recognition due to their brightness, photostability, and multiplexing capability.36 Principle of the multiplexed imaging and pattern recognition is illustrated in Figure 1. QDs with three different fluorescence emission wavelengths were imprinted on their surface using SA, Fuc, and Man as the template, respectively. Through simultaneously staining the cells under investigation with the SA-, Fuc-, and Man-imprinted QDs, multiplexed imaging of the cells can provide information on the expression levels of the three template monosaccharides on the cells. The fluorescence intensities for the three monosaccharides on different types of cells were read out to construct pattern
ecognition of cancer cells is essential for many important areas such as cancer diagnosis and targeted cancer therapy.1−3 Differences in the structure and properties of compounds expressed on the surface of cells, such as cancerspecific antigens, have been an important basis for the screening or preparation of cancer cell-targeting reagents. A variety of strategies have been employed for the recognition of cancer cells, such as immune recognition,4−6 aptamer-matching,7−9 and ligand−receptor interaction.10,11 These strategies, however, are constrained by the rarity of cancer-specific single species. To this end, multitarget-based recognition can be a useful strategy to improve the recognition specificity. On the other hand, recognition of a specific cell type among many cell types is critical in important fields, such as cell sorting and cancer diagnosis. Such specific recognition requires a sound understanding of the structure, nature, and expression of species that can function as targets for the recognition. Aberrant expression of glycan structures on cell surface is a universal hallmark of cancer cells. For instance, sialic acid (SA)12,13 and fucose (Fuc)14,15 are overexpressed on most cancers, while mannose (Man) is overexpressed on certain cancers such as liver cancer.16,17 Clearly, the combination of multiple monosaccharides could be effective for specific cancer recognition. However, to the best of our knowledge, such a possibility has not been explored yet. Molecularly imprinted polymers (MIPs),18−21 as antibody mimics synthesized through polymerization in the presence of a template, exhibit affinity and specificity to the template. As compared with antibodies, MIPs are easy to prepare, costefficient, and more stable. MIPs have found important applications in many areas, such as chemical sensing,22,23 separation,24,25 and disease diagnostics.26,27 Recently, MIPs © XXXX American Chemical Society
Received: March 15, 2017 Accepted: April 25, 2017 Published: April 25, 2017 A
DOI: 10.1021/acs.analchem.7b00965 Anal. Chem. XXXX, XXX, XXX−XXX
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Figure 1. Schematic of pattern recognition of cells via multiplexed imaging with monosaccharide-imprinted QDs.
Apparatus. Transmission electron microscopy (TEM) was carried on a JEOL JEM-1011 instrument (Tokyo, Japan). Fluorescence properties of the prepared QDs and evaluation of boronic acid functionalization as well as molecular imprinting were performed on a Synergy Mx microplate reader from BioTek (Winooski, VT, U.S.A.). Fourier transform-infrared (FT-IR) characterization was performed on a Nicolet iS10 instrument (Thermo Fisher Scientific, Shanghai, China). Cell imaging was performed on a Leica TCS SP5 laser scanning confocal microscope (Leica Microsystems CMS GmbH, Mannheim, Germany) with an objective lens (×60). Preparation and Boronic Acid-Functionalization of Carboxyl-Capped CdTe QDs. Water-dispersible carboxylcapped CdTe QDs were prepared according to a literature method37 with slight modifications. Briefly, 91.34 mg of CdCl2· 2.5H2O was first dissolved into 100 mL of water, followed by adding with 36 μL of TGA and the solution pH was adjusted to 10.8 using 0.1 M NaOH. Then, the mixture solution was kept in vigorous stirring for 5 min. After that, 100 mL of water containing 19.72 mg of Na2TeO3 was supplemented to the mixture solution, the obtained solution was continuously stirred for another 5 min and the solution pH was kept in the range of 10.5−11.0 during this period. Then, 160 mg of NaBH4 was added and the obtained solution was ultrasonicated for 3 min. Finally, the resulted solution was refluxed in oil bath at 120 °C for an appropriate duration. Fluorescence emission spectrum of this solution was monitored every 15 min until the desired emission wavelength appeared (525, 565, and 605 nm). Carboxyl-capped QDs were separated via centrifuging the precipitation generated by excessive absolute ethanol, which quadruples the QDs solution in volume. The synthesized QDs were washed with ethanol for three times and dried in vacuum oven at 45 °C. The boronic acid-functionalization of carboxyl-capped QDs was carried out according to a literature method38 with slight modifications and the synthesis route is shown in Figure S1A. Briefly, 20 mg of QDs was first dispersed into 20 mL of 10 mM phosphate buffer (pH 7.4), then the solution was ultrasonicated for 5 min. After that, 27 mg of EDC and 14 mg of NHS were added into the solution. After ultrasonicated for 30 s, the obtained solution was vigorously stirred for 2 h in ice bath. After that, 27 mg of APBA was added and the solution was continuously stirred for another 2 h in ice bath and then kept at room temperature for 24 h. After the reaction had finished, the solution was centrifuged at 5000 rpm for 5 min. After removing the unreacted reagents, 80 mL of ethanol was added into the transparent supernatant and the solution was kept for several minutes until flocculent precipitate appeared. Finally, boronic acid-functionalized QDs were acquired by centrifuging at 8,000 rpm for 5 min. The resulted QDs were washed with absolute ethanol for three times and then dried in vacuum oven at 45 °C.
recognition. Seven cancerous cell lines and four normal cell lines were used as the model cell lines. The multiplexed imaging-based pattern recognition revealed not only the relative expression levels of the monosaccharides on the 11 cell lines, but also disclosed the similarities and differences of the cell lines. It allowed for not only the recognition of cancer cells from normal cells, but also the recognition of specific cancer cells.
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EXPERIMENTAL SECTION Materials and Chemicals. CdCl 2 ·2.5H 2 O (99.9%), Na2TeO3 (99.9%), thioglycolic acid (TGA, 98%), 1-(3(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC, 98.5%), D-fucose (Fuc, 99%), and D-N-acetylneuraminic acid (SA, 98%) were purchased from Aladdin Industrial (Shanghai, China). γ-Aminopropyltriethoxysilane (APTES, 98%), N-hydroxysuccinimide (NHS, 98%), formylphenylboronic acid (FPBA, 97%), NaBH3CN (95%), D-galactose (Gal, 98%), and D-glucose (Glc, 99%) were purchased from J&K Chemical (Shanghai, China). D-Mannose (Man, 99%) was purchased from Alfar Aesar (Tianjin, China). Ammonia−water (28%) and acetic acid (HAc, AR) were purchased from Sinopharm Chemical Reagent (Shanghai, China). NaBH4 (98%) was purchased from Macklin (Shanghai, China). Tetraethyl orthosilicate (TEOS, 99%) was purchased from Heowns Biochemical Technology (Tianjin, China). 3-Aminophenylboronic acid (APBA, 98%) was purchased from Energy Chemical (Shanghai, China). Hepatoma carcinoma cell (HepG2), normal hepatocyte cell (L-02), mammary cancer cell (MCF7), normal mammary epithelial cell (MCF-10A), human epidermal carcinoma cell (A-431), normal epidermal cell (HaCat), renal carcinoma cell (OS-RC-2), normal renal epithelial cell (HK-2), gastric carcinoma cell (SGC-7901), colorectal carcinoma cell (HCT-8), human cervical carcinoma cell (HeLa), phosphate buffer solution for cell culture (1× PBS), parenzyme cell digestion solution (containing 0.25% trypsin and 0.02% EDTA), Dulbecco Modified Eagle Medium (DMEM, containing 4.5 mg/mL D-glucose, 80 U/mL penicillin, and 0.08 mg/mL streptomycin), Roswell Park Memorial Institute 1640 medium (RPMI-1640, containing 2.0 mg/mL D-glucose, 0.3 mg/ mL glutamine, 2.0 mg/mL NaHCO3, 80 U/mL penicillin, and 0.08 mg/mL streptomycin), and calf serum were purchased from Keygen Biotech (Nanjing, China). Fetal bovine serum (FBS) was purchased from Gibco (Life Technologies, Australia). Horse serum was purchased from Sigma-Aldrich (Shanghai, China). All other chemical reagents were of analytical grade unless otherwise noted. Water used in all experiments was purified by a Milli-Q Advantage A10 ultrapure water purification system (Millipore, Milford, MA, U.S.A.). Cell culture bottle (25 cm2 in growth area) and glass bottom cell culture dishes (Φ 30 mm) obtained from NEST Biotechnology (Wuxi, China) were used for cell culture and imaging. B
DOI: 10.1021/acs.analchem.7b00965 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry Preparation of Monosaccharide-Imprinted QDs. Monosaccharide-imprinted QDs were prepared according to the boronate affinity oriented surface imprinting approach,30 which included three steps: (1) template immobilization, (2) oriented imprinting, and (3) template removal. The synthesis route of monosaccharide-imprinted QDs is shown in Figure S1. For immobilization of the template on boronic acid-functionalized QDs, 40 mg of monosaccharide template was added into 20 mL of phosphate buffer (0.1 M, pH 7.4) containing 1 mg/mL boronic acid-functionalized QDs and the pH was adjusted to 7.4. After incubation for 30 min, 80 mL of ethanol was added into the solution and set for several minutes until flocculent precipitate arrived, then the monosaccharide-bound QDs were collected via centrifuging at 8000 rpm for 3 min, followed by washing with 0.1 M phosphate buffer (pH 7.4) for twice. QDs with emission wavelength of 525, 565, and 605 nm were used to bind with Man, SA, and Fuc, respectively. The above-mentioned monosaccharide-bound QDs were collected and redispersed into 20 mL of ethanol, added with 0.35 mL of ammonia and 5 mL of prepolymer solution that contained 22.4 μL of TEOS and 10 mL of ethanol. After reaction for an appropriate duration, the mixture was centrifuged and the precipitates were collected. To remove the template from the imprinted QDs, the collected precipitates were washed with 0.1 M HAc for 3 h, followed with 0.1 M phosphate buffer (pH 7.4) for 30 min and centrifugation again. The obtained monosaccharide-imprinted QDs were collected and stored in 1× PBS. To prepare nonimprinted QDs for comparison, the procedures were the same except that no template was immobilized onto boronic acid-functionalized QDs. Imprinting Factor. The monosaccharide-imprinted and nonimprinted QDs were assessed in terms of imprinting factor via the boronate affinity sandwich assay method27 using template molecule as a bridge molecule. The amount of 2 mL of monosaccharide-imprinted and nonimprinted QDs solution (1 mg/mL for each) were added with template molecule (final concentration, 2 mg/mL) and the pH was adjusted to 7.4. After incubation for 30 min, ethanol quadrupled the QDs solution in volume was added into each QDs solution and then set for several minutes until flocculent precipitate appeared. Then the precipitation was collected by centrifuging at 8000 rpm for 3 min and rinsed with 0.1 M phosphate buffer (pH 7.4) for twice, and then redispersed into 2 mL of 0.1 M phosphate buffer (pH 7.4). After that, the template-bound imprinted QDs solution and nonimprinted QDs solution were, respectively, added into a 3 × 4 array of boronic acid-functionalized 96-well microplate (250 μL for each well), and an equal volume of 0.1 M phosphate buffer (pH 7.4) without any QDs and templates was added into the array as control. After incubation for 30 min, the array was washed with 0.1 M phosphate buffer (pH 7.4) three times. Finally, fluorescence of the resultant array was read on the microplate reader and the fluorescence intensity of imprinted QDs and nonimprinted QDs was control-subtracted and averaged over column. The IF values were calculated by dividing the fluorescence intensity of imprinted QDs by that of nonimprinted QDs. Selectivity Test. A 6 × 4 array of a boronic acidfunctionalized 96-well microplate was used for the experiments. The array was added with equivalent volumes of six different solutions and incubated for 30 min: (A1−D1) 0.1 M phosphate buffer (pH 7.4) containing 2 mg/mL Gal; (A2−D2) 0.1 M phosphate buffer (pH 7.4) containing 2 mg/mL Fuc; (A3−D3)
0.1 M phosphate buffer (pH 7.4) containing 2 mg/mL Glc; (A4− D4) 0.1 M phosphate buffer (pH 7.4) containing 2 mg/mL Man; (A5−D5) 0.1 M phosphate buffer (pH 7.4) containing 2 mg/mL SA; and (A6−D6) 0.1 M phosphate buffer (pH 7.4) without any additives (used as control). After incubation, the array was washed with 0.1 M phosphate buffer (pH 7.4) three times and then added with equivalent volume of 0.1 M phosphate buffer (pH 7.4) containing 1 mg/mL monosaccharide-imprinted QDs and incubated for another 30 min. Thereafter, the array was washed with 0.1 M phosphate buffer (pH 7.4) three times. Finally, fluorescence of the array was read on the microplate reader and the fluorescence intensity was control-subtracted and averaged over each column. Cell Culture, Imaging, and Intensity Readout. HepG-2, L-02, MCF-10A, A-431, HaCat, HK-2, and HeLa cells were cultured in the DMEM medium with 10% fetal bovine serum for 2 to 3 days (37 °C, 5% CO2); MCF-7 and OS-RC-2 cells were cultured in the RPMI-1640 medium with 10% fetal bovine serum for 3 to 4 days (37 °C, 5% CO2); SGC-7901 cell was cultured in RPMI-1640 medium with 10% calf serum for 2 to 3 days (37 °C, 5% CO2); HCT-8 cell was cultured in RPMI-1640 medium with 10% horse serum for 2 to 3 days (37 °C, 5% CO2). The cell culture medium was removed when the confluence up to 70− 80% and the cells remained in culture dishes were washed with 1× PBS for three times. Then the cells were incubated with 1 mL of 1× PBS solution containing SA-, Fuc- and Man-imprinted QDs (200 μg/mL each) for 30 min. The cells incubated with 1 mL of 1× PBS solution containing nonimprinted QDs (200 μg/ mL) were used as control. The PBS buffer and free QDs were removed and the acquired cells were rinsed with 1× PBS for three times again and supplemented with 1 mL of 1× PBS. Multiplexed fluorescent images were obtained on a laser scanning confocal microscope with a sequence mode. The excitation wavelength was set at 405 nm for all imaging processes. From the obtained images, totally 50 cells with representative shape and size for each cell line were selected for intensity readout, then the fluorescence intensity of the selected 50 cells was averaged. The pattern recognition was constructed on the basis of the distinction in fluorescence intensities of different cell lines for each type of the imprinted QDs. Establishment of Pattern Recognition. Three pattern recognition techniques, including spatial distribution analysis, principal component analysis (PCA) and cluster analysis, were employed to construct diagrams for pattern recognition. For spatial distribution analysis, a 3D plot was drawn using the fluorescence intensity at each emission wavelength directly as a coordinate axis. For PCA and cluster analysis, the data were processed using the Multivariate Analysis function of the software OriginPro 2017C (64-bit) (OriginLab Corporation, Northampton, MA). Treatment of Cells by Glycosidases. The cell culture medium was removed at first, then the cells remained in culture dish were washed with 1× PBS for three times. The obtained cells followed by incubating with culture medium containing sialidase, fucosidase, and mannosidase (1 unit/mL for each) for 4 h. After that, the culture medium was removed and the resulted cells were washed with 1× PBS for three times again. Finally, the sialidase-, fucosidase-, and mannosidase-treated cells were stained with SA-, Fuc-, and Man-imprinted QDs and followed by fluorescence imaging as the procedures described above. C
DOI: 10.1021/acs.analchem.7b00965 Anal. Chem. XXXX, XXX, XXX−XXX
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Figure 2. Selectivity of SA- (A), Fuc- (B), and Man-imprinted (C) QDs toward monosaccharides evaluated via boronate affinity sandwich assay.
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RESULTS AND DISCUSSION Characterization of Monosaccharide-Imprinted QDs. The synthesis route of the monosaccharide-imprinted QDs is illustrated in Figure S1. Carboxyl-capped CdTe QDs were selected as the core for the fluorescent nanoprobes due to their excellent water dispersity and feasible surface chemistry. The prepared QDs showed multicolor and tunable emission wavelength (Figure S2). The QDs were functionalized with boronic acid via amidation reaction between the carboxyl group of the QDs and the amino group of 3-aminophenylboronic acid (APBA). Successful conjugation of APBA onto the QDs was confirmed by FT-IR spectroscopy. As shown in Figure S3, the strong bands at 1665 and 1578 cm−1 indicate the formation of amido bond, while the characteristic band of boronic acid at 1327 cm−1 and the out-of-plane deformation vibration of substituted benzene ring at 709 cm−1 confirm the existence of APBA. Moreover, the boronate affinity sandwich assay27 was used to evaluate the boronic acid-functionalization. The boronic acidfunctionalized QDs exhibited apparent boronate affinity toward glucose (Figure S4), suggesting the QDs were successfully conjugated with APBA. Transmission electron microscopy (TEM) characterization suggests that both carboxyl-capped and imprinted QDs possessed uniform morphology and excellent water dispersity (Figure S5). The particle size of carboxyl-capped QDs was approximately 5−8 nm (Figure S5A), a thin imprinting layer on the monosaccharide-imprinted QDs was observed and its thickness was roughly estimated to be 1−2 nm (Figure S5B). The imprinted QDs showed similar excitation band but strong and different emission bands, located at 525, 565, and 605 nm, respectively (Figure S6). As compared with the carboxyl-capped QDs, the monosaccharide-imprinted QDs exhibited lighter color due to the presence of the imprinting layer, but the fluorescence brightness of monosaccharide-imprinted QDs was nearly the
same under UV irradiation (Figure S7). All of these results confirm the feasibility of using monosaccharide-imprinted QDs for multiplexed imaging. The molecular imprinting was carried out according to the boronate affinity oriented surface imprinting approach.30 The imprinting time was individually set at 20, 15, and 15 min for imprinting of SA, Fuc, and Man, giving an imprinting factor of 7.9, 14.9 and 11.7, respectively (Figure S8). Such values are well acceptable in molecular imprinting. Moreover, the monosaccharide-imprinted QDs exhibited good specificity toward the target monosaccharides, with cross-reactivity toward nontarget monosaccharides less than 23.5, 26.7 and 27.6% for SA-, Manand Fuc-imprinted QDs, respectively (Figure 2). Larger crossreactivity of Man- and Fuc-imprinted QDs was due to the structural similarity of Fuc and Man. It was observed in Figure 2B that the binding of nonimprinted QDs toward some nontarget monosaccharides was higher than that of Fuc-imprinted QDs, which was unexpected. Such abnormal phenomenon was repeatable, but the reason is unclear at present. Multiplexed Cell Imaging via Monosaccharide-Imprinted QDs. Seven cancerous cell lines (including HepG-2, MCF-7, OS-RC-2, A-431, HCT-8, HeLa, and SGC-7901) and four normal cell lines (including MCF-10A, L-02, HK-2 and HaCat) were chosen as model cell lines. As shown in Figures 3 and S9, after simultaneously stained with Man-, SA- and Fucimprinted QDs, all the normal cell lines showed only very weak fluorescence. In contrast, all the cancer cell lines exhibited stronger fluorescence and their shapes were apparently fluorescently displayed in mono- or multicolor. As a control, after incubation with nonimprinted QDs, all cancer cells exhibited almost no fluorescence (Figure S10). These results indicate that the targeting of cancer cells by the monosaccharideimprinted QDs was due to the specific recognition of imprinted cavities rather than nonspecific interactions. D
DOI: 10.1021/acs.analchem.7b00965 Anal. Chem. XXXX, XXX, XXX−XXX
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Figure 3. Confocal imaging of human carcinoma cells via monosaccharide-imprinted QDs. (A) MCF-7 (breast carcinoma cell); (B) SGC-7901 (gastric carcinoma cell); (C) HepG-2 (hepatoma carcinoma cell); (D) OS-RC-2 (renal carcinoma cell); (E) HeLa (cervical cancer cell); (F) A-431 (epidermal carcinoma cell); (G) HCT-8 (colon carcinoma cells). Columns from left to right: dark field of Man-, SA-, and Fuc-imprinted QDs stained cells and overlaid field of bright field and multiplexed dark fields.
The relative expression of the template monosaccharides on different cancerous cell lines was evaluated in terms of the fluorescence intensity. After simultaneously stained with the
three types of monosaccharide-imprinted QDs, MCF-7 cells exhibited comparable expression level of SA and Fuc, while the expression level of Man was much lower; SGC-7901 cells E
DOI: 10.1021/acs.analchem.7b00965 Anal. Chem. XXXX, XXX, XXX−XXX
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more than 91% (Figure 5), indicating that the 2D PCA could cover and express the correlation of the analyzed object to a large
displayed high expression level of SA while the expression levels of Man and Fuc were much lower; HepG-2 cells exhibited comparable expression level of Man, SA and Fuc; OS-RC-2 and HeLa, A-431, and HCT-8 cells showed comparable expression level of Man and SA, but the expression level of Fuc was much lower (Figure 3). These results suggest that MIPs-based multiplexed imaging could be a useful means for the differentiation of cancer cell lines. Specificity Verification by Glycosidase Treatment. The specificity of the monosaccharide-imprinted QDs toward the target monosaccharides was further confirmed by glycosidasetreated cell imaging. Sialidase, fucosidase, and mannosidase was used for cutting off SA, Fuc, and Man from the cells, respectively. OS-RC-2, HepG-2, MCF-7, and SGC-7901 cells were used as model cancer cells for this verification. All the cells exhibited very weak fluorescence after the target monosaccharides on the cell surface were removed, regardless of which cell line and imprinted QDs used (Figure S11). These results suggest that the multiplexed cell imaging of the monosaccharide-imprinted QDs was due to their specific binding with the target monosaccharides. Spatial Distribution Analysis. After being simultaneously stained with the monosaccharide-imprinted QDs, the 11 cell lines exhibited largely varied intensities (Figure S12), which indicate distinct expression levels of the three monosaccharides on cells of different cell lines. Since spatial distribution analysis uses the signal intensity directly as coordinate axes and avoids complicated mathematical processing (though it can be performed on commercial software), it was first carried out for pattern recognition. As shown in Figure 4, a 3D plot was obtained
Figure 5. Principal component analysis of the 11 different cell lines.
extent. The PCA diagram indicates that the cancerous cell lines are discretely distributed in their feature spaces, while all the normal cell lines are located in a compact common region; that is to say, cancerous cell lines could be distinguished from each other according to their feature space, while different normal cell lines are hard to differentiate. Although A-431 and HCT-8 cells are shown to be highly overlapped in the 3D spatial distribution analysis in Figure 4, they are disclosed to be only partially overlapped in the 2D PCA in Figure 5. This suggests that, if constructed reasonably, PCA could provide clearer recognition toward cells with highly similar monosaccharide expression than spatial distribution analysis. Therefore, the PCA analysis established herein is an effective tool for the recognition of different cell lines. Cluster Analysis. Cluster analysis was performed to further disclose the similarities of different cell lines. In this technique, cell lines with more similar monosaccharide expression tend to be clustered apart from less similar ones; in other words, the cluster is in a step-by-step hierarchical pattern. As shown in Figure 6, cell line HepG-2 and MCF-7, HCT-8 and A-431, OSRC-2 and SGC-7901, L-02 plus MCF-10A and HK-2 best match at the first level, which suggests that the members in these groups are the most similar; at the second level, HaCat is classified into the L-02 plus MCF-10A and HK-2 group, while the HCT-8 and A-431 pair converge with the HaCat, L-02, MCF-10A, and HK-2 group in the next level, and a higher level means less similarity.
Figure 4. Spatial distribution of the 11 cell lines constructed by multiplexed imaging via simultaneously staining with Man-, SA-, and Fuc-imprinted QDs.
by spatial distribution analysis, which intuitively shows the spatial distributions of these cell lines in the coordinate system. The spatial distributions of normal cell lines are highly centralized and very close to the origin of the coordinate system, which reveals that the expression levels of the three monosaccharides of normal cell lines are all very low. As a comparison, the cancer cell lines are located in spaces much farther from normal cell lines and most of them much more scattered from each other, except A-431 and HCT-8, which are closely neighboring. This suggests that most cancer cell lines are much diverse in their monosaccharide expression. PCA Analysis. PCA was then established on the basis of the fluorescence intensities of different cell lines for each type of the imprinted QDs. The first two PCA components totally occupy
Figure 6. Cluster analysis of the 11 different cell lines. F
DOI: 10.1021/acs.analchem.7b00965 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry
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Different from the 3D spatial distribution analysis and the PCA analysis discussed above, the cluster analysis can qualitatively indicate the inter-relation in the expression level of the three monosaccharides on different cell lines and, thus, disclose a more close relationship between different cell lines. Since the three pattern recognition techniques have different strengths, they can be combined into an efficient toolbox for the recognition of cells.
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CONCLUSIONS In summary, we have prepared three types of monosaccharideimprinted QDs for the multiplexed imaging and pattern recognition of cells. Multiplexed imaging of cells simultaneously stained with the monosaccharide-imprinted QDs revealed the relative expression levels of these monosaccharides on the cells. Pattern recognition constructed using the fluorescence intensities of multiplexed imaging disclosed the similarities and differences of different cell lines, allowing for not only the recognition of cancer cells from normal cells, but also the recognition of specific cancer cells. Thus, this study paved a solid ground for the design and preparation of novel cancer-cell targeting reagents and nanoprobes.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b00965. Additional experimental data (PDF).
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AUTHOR INFORMATION
Corresponding Author
*Tel.: +86 25 8368 5639. Fax: +86 25 8368 5639. E-mail:
[email protected]. ORCID
Zhen Liu: 0000-0002-8440-2554 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge financial support from the National Science Fund for Distinguished Young Scholars (No. 21425520) and the Instrumentation Grant (No. 21627810) from the National Natural Science Foundation of China, the “333” Talents Project from Jiangsu Provincial Government (No. BRA2016351), and the Open Grant from the State Key Laboratory of Analytical Chemistry for Life Science (No. 5431ZZXM1605).
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DOI: 10.1021/acs.analchem.7b00965 Anal. Chem. XXXX, XXX, XXX−XXX
