Visual detection of cancer cells by colorimetric aptasensor based onaggregation of gold nanoparticles induced by DNA hybridization
Yasaman-Sadat Borghei a, Morteza Hosseini a, *, Mehdi Dadmehr b, Saman Hosseinkhani c,Mohammad Reza Ganjali d, e, Reza Sheikhnejad f
a Department of Life Science Engineering, Faculty of New Sciences & Technologies, University of Tehran, Tehran, Iranb Payame Noor University, Tehran, Iranc Department of Biochemistry, Tarbiat Modares University, Tehran, Irand Center of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, Tehran, Irane Biosensor Research Center, Endocrinology & Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iranf Department of Molecular Biology, Tofigh Daru Co., Tehran, Iran
A simple but highly sensitive colorimetric method was developed to detect cancer cells based on aptamerecell interaction. Cancer cells were able to capture nucleolin aptamers (AS 1411) through affinityinteraction between AS 1411 and nucleolin receptors that are over expressed in cancer cells, The specificbinding of AS 1411 to the target cells triggered the removal of aptamers from the solution. Therefore noaptamer remained in the solution to hybridize with complementary ssDNA-AuNP probes as a result thesolution color is red. In the absence of target cells or the presence of normal cells, ssDNA-AuNP probesand aptamers were coexisted in solution and the aptamers assembled DNA-AuNPs, produced a purplesolution. UVevis spectrometry demonstrated that this hybridization-based method exhibited selectivecolorimetric responses to the presence or absence of target cells, which is detectable with naked eye. Thelinear response for MCF-7 cells in a concentration range from 10 to 105 cells was obtained with adetection limit of 10 cells. The proposed method could be extended to detect other cells and showedpotential applications in cancer cell detection and early cancer diagnosis.
measuring the number of specific cells in human blood, clinicianscould determine the onset of a specific disease or predict the pa-tient's response to a specific treatment. The ability to detect specificcells at a very low concentration is critical for detecting circulatingtumor cells. Because most solid tumor cells can be found at aconcentrations up to 200 cells/ml in an average adult male (0.004%of cell population in the blood) [1]. More importantly, screeningpeople with no symptoms to find early signs of cancer in a routineblood test would increase the chances of being cured dramatically[2,3]. However, traditional analysis techniques, such as immuno-histochemistry, flow cytometry, and polymerase chain reaction(PCR) [4e6] do not meet the requirement for point-of-care (POC)diagnostics because they usually require costly instruments, longanalytical time, and complicated operations [7]. Meanwhile,considering the very low quantity of stray cancer cells [1,8],screening requires a new clinical platformwith high specificity andultra-sensitivity to detect cancer cells, especially for early clinicaldiagnostics. Furthermore, the screening approach should beaffordable to screen more people, especially in developing coun-tries and detect cancer at early stages that would increase thesurvival rate tremendously. Therefore, there is a need for devel-oping a simple, sensitive and affordable diagnostic tool to detectrare circulating cancer cells.
Coupling nanomaterials and biomolecule recognition eventsrepresents a new direction toward the development of novel mo-lecular diagnostic tools [9]. For detection of cancer cells, not only itis important to have a specific platform, but we also need to have ahighly sensitive tool as well. The advanced detection techniquesAptamers designed allow us to design aptamers with predictablestructures and site-specific chemical modification to provide link-age for advanced signaling mechanism. AS1411 (26 mer, 7.8 kDa) isa GC-rich DNA aptamer that binds to nucleolin with a high affinity(dissociation constant is in the picomolar to low nanomolar range)[10,11]. Nucleolin is a phosphoprotein overexpressed in cytoplasmand on plasma membrane of the metastatic cells, and not thenormal cells. AS1411 enters many different cancer cell-types vianucleolin-mediated endocytosis [12,13].
Aptamers were conjugated with nanomaterials to enhancecancer cell detection. For instance, the density of cell-surface tar-gets for aptamers is not always abundant, especially for cancer cellsin the early stages of development. Therefore, multivalent bindingversus single aptamer binding has been studied to increase cellspecific signaling. The large surface area and variable sizes allownanomaterials to serve as multivalent ligand scaffolds [14,15]. Tomake the assay colorimetric, gold nanoparticles were utilizedbecause of their bio functionalization, bio stability, and spectralproperties. Due to the plasmon resonance of gold nanoparticles,they possess strong distance-dependent optical properties. Oncethe gold nanoparticles come into proximity with one another, theirabsorption spectra shift and their scattering profile changes resultchanges in color and absorption spectra of the sample [15,16]. As aresult, many techniques have been developed based on goldnanoparticles aggregation to detect ions, genes and proteins[17e21]. Considering unique properties of the aptamers and goldnanoparticles, we have developed an assay system that is colori-metric in nature and shows excellent selectivity between target andcontrol cells. This method is based on hybridization betweenaptamer and two sets of gold nanoparticles functionalized withsingle-stranded DNA probes in supernatant (probe 1 and 2 e
AuNPs). In this study since both probes had partially complemen-tary sequences to specific site of aptamer, cross-linking of nano-particles was induced by hybridization under target cell freecondition. This phenomenon resulted in gold nanoparticle aggre-gation and produced a purple solution. In the presence of targetcells, the specific binding of AS 1411 to the target cells triggered
aptamer removal from solution, no aptamer was remained in thesolution to assemble DNA-AuNPs aggregation that resulted a redcolor solution.
2. Materials and methods
2.1. Materials and reagents
Chloroauric acid (HAuCl4_4H2O) and sodium citrate(Na3C6H5O7) were purchased from Sigma-Aldrich. Sodium hy-droxide (NaOH) and hydrochloric acid (HCl) were obtained fromMerk. Fetal bovine serum (FBS), Dulbecco's modified eagle medium(DMEM), and penicillin/streptomycin were purchased from Gibco(USA). Deionized water with a resistivity greater than 18 MU cmwas acquired from a Millipore Milli-Q system. Phosphate-bufferedsaline (PBS) was prepared by mixing 8 g NaCl, 0.2 g KCl, 1.42 gNa2HPO4 and 0.27 g KH2PO4 in 1 L of twice distilled water. All oli-gonucleotides used in this work were synthesized by ShanghaiGeneray BiotechCo. Their bases sequences are shown as follows:
All oligonucleotides stock solutions were prepared with TEBuffer and kept frozen until used. To make a TE Buffer, 1 ml of 1 MTriseHCl (pH7.5) and 0.2 ml EDTA (0.5 M) was added to deionizedwater to a total volume of 100 ml of solution. All chemicals were ofanalytical grade and used without further purification.
Cells and cell culture human cell lines used in this study wereMCF-7 cells (human breast cancer cell line), A549 cells (human lungcancer cell line), AGS cells (human gastric cancer cell line), andprimary fibroblast cells (from normal human skin).
2.2. Apparatus
Absorption spectra were determined using PerkineElmerlambda25 spectrometer. TEM images were taken with a trans-mission electron microscope (Zeiss, EM10C, 80 KV, Germany) on acopper grid.
2.3. Synthesis of gold nanoparticles
Fifty ml aqueous solution of hydrogen tetrachloroaurate (III)tetrahydrate (1 mM) was heated to boiling while being stirred in around-bottom flask with a reflux condenser. 10 ml of trisodiumcitrate (38.8 mM) was then added into the solution rapidly. Thesolution was boiled again for another 10 min and the color of thesolution changed from yellow to red. The heating was stopped butthe stirring continued until it reached room temperature [22], TheAuNPs solution was then stored in at 4 �C. The TEM imaging anal-ysis determined the diameter and dispersion state of our synthe-sized AuNPs. Using the extinction coefficient (2.7 � 108 M�1 cm�1)at 520 nm [23], the concentration of the AuNPs solution wascalculated to be about 4.4 nM according to Beer's law. The diameterof the synthesized AuNPs was about 25 nm.
2.4. Preparation of DNA-Modified AuNPs
The coupling of thiolated probe to gold nanoparticles wasdemonstrated by the higher affinity of thiol to AuNPs. Thiolatedprobe (1 OD) and 1 ml of the gold nanoparticles solution wasincubated at room temperature for 16 h [24]. The solution wastransferred into 0.1 M NaCl, 10 mM phosphate buffer (pH 7) andkept at room temperature for 40 h. To remove unreacted probes,
the solution was centrifuged at 14,000 rpm for 25 min. The su-pernatant was removed and the pellet was resuspended in 1 ml ofthe same buffer and stored at 4 �C until used.
2.5. Cell culture
MCF-7 cells (human breast cancer cell line), A549 cells (humanlung cancer cell line), AGS cells (human gastric cancer cell line),primary fibroblast cells (from normal human skin) were cultured in25 cm2 tissue culture flasks (SPL, Korea) with 6 ml Dulbecco'smodified Eagle's medium (Sigma, UK) containing 10% heat-inactivated fetal bovine serum (Gibco), 100 IU/ml penicillin and100 mg/ml streptomycin (Sigma, UK). Cells were incubated under95% air, 5% CO2 in a humidified incubator for 6 days until the cellmonolayer became confluent. Growth medium was replaced withfresh media every 2 days or as required, indicated by color changedue to low pH. Upon reaching at least 80% confluence, the cellswerewashedwith phosphate-buffered saline (PBS) and trypsinizedfor 10 min at 37 �C with 0.05% trypsin and 0.02% ethylenediaminetetraacetic acid.
2.6. Incubation of aptamer with cancerous and normal cells
The cell number was determined using a hemocytometer priorto in vitro assay performance. 106 cells were dispersed in DMEMcell culturemedia and centrifuged at 1000 rpm for 10 min. Thewashing was repeated two more times and finally the cells wereredispersed in 100 mL of buffered media. In the next step, 10 ml ofaptamer (1 mM and 10 mM) was added to the 90 mL of 10 mMphosphate buffer saline (PBS) [25]. After 1hr incubation, the cellswere centrifuged at 1000 rpm for 10 min and the supernatant wastransferred to microtubes for hybridization process (Scheme 1).
Scheme 1. Schematic representation of selective colorimetric method for detection of canaptamer.
2.7. Hybridization in supernatant
10 ml of the suspension containing functionalized nanoparticleprobes (1&2) were added to supernatant. To improve the hybridi-zation efficiency, themixturewas incubated at 95 �C for 10min. Thehybridization process was then carried out by gentle stirring at37 �C (Scheme 2).
2.8. Optical detection
The colorimetric detection of cancer cells was performed asfollowing: The aptamer was incubated with different number ofcells at 37 �C for 1 h. Then suspension was centrifuged pellet wasremoved and the solution containing probe1-AuNPs and probe2-AuNPs was added to supernatant to allow assembly of the DNA-AuNPs.
Hybridization efficiency between two types of probe-AuNPs andaptamers depend on the amount of aptamers remained in super-natant (which represents the number of cells or the cell surfacereceptor expression) (Scheme 2). The absorbance was measured at400 nme800 nm wavelength range.
3. Results and discussion
3.1. Principle of the aptamer based colorimetric method
The principle of the colorimetric assay for detection of cancercells based on aptamer and two sets of DNA-modified AuNPs ispresented in Scheme 1. The specific binding of the AS1411 aptamerto cancer cells lead to the removal of aptamer from the solution. Inour detection system aptamer play a role as a linker DNAwhich hascomplementary sites with each of two sets of probes-modifiedAuNPs.
When the aptamer was incubated with MCF-7 cells, the bindingof the aptamer to the cell membrane was facilitated due to its
cer cells by employing DNA probe 1,2 -functionalized gold nanoparticles and AS1411
Scheme 2. Comparing the efficiency of hybridization aptamer with increasing of concentration of aptamer: (a) blank, (b) the lower concentration than probe, (c) a concentrationequal to probe.
affinity for protein receptors (nucleolin), leading to the uptake ofaptamer from the solution. Therefore aptamer could no longerassemble DNA-AuNPs dimer (Scheme 1) and the red color ofseparated AuNPs was observed. By increasing the number of MCF-7cells, more linker DNA (aptamer) was removed, andmore separated(rather than aggregated) AuNPs were present in the solution. Thusthe target cells were detected qualtitatively and visually by ourAuNP-based colorimetric aptasensor (Scheme 2).
Fig. 1. Signaling profile of the system using different concentration of aptamer inpresence of MCF-7 cells (1.0 � 105, 104, 103, 102, 10 cells/ml MCF-7) and in the absenceof MCF-7 cells (as a negative control).
3.2. Characterization of probe1,2/Apt/AuNPs
The shape and size of the nanoparticles were determined byTEM imaging of the nanoparticles before and after hybridization.The nanoparticles appeared nearly spherical and had an averagediameter of about 25 nm. To further confirm that the hybridizationcan alter the optical properties of DNA-modified AuNPs, TEM im-aging was used to test the feasibility of our colorimetric design.Scheme 2 shows the color changes and aggregation analysis ofdifferent supernatant with different color changes. As we expected,by increasing the number of MCF-7 cells, more aptamers wereremoved from suspension due to cellular uptake., Therefore hy-bridization did not occurred between DNAand AuNPs and a redcolor solutionwas observed (Scheme 2A). This implies that probe1-AuNPs and probe2-AuNPs did not assemble. But at lower concen-tration of MCF-7 cells, less aptamers were removed from suspen-sion therefore purple color solutions were observed and this colorchange represents the aptamer-induced aggregation of AuNPs(Scheme 2B). On the other hand, in the absence of cancer cells,MCF-7 or in the presence of normal cells, it produced a visual colorchange and the absorption peak shift (Scheme 2C). Therefore, thisratio (aptamers remained in supernatant to probe1,2-AuNPs) wasused for quantitative analysis of cells; a low ratio is represented byred color solution (dispersed DNA-modified AuNPs), and a highratio is represented by purple-colored solution (aggregates).
3.3. Influence of different concentration of the aptamer
In order to achieve the optimal cell detection condition, theexperiment was carried out at different concentration of aptamers(0, 1 mM, 10 mM and 100 mM). The results shown in Fig. 1, indicatethat, significant absorbance variations were observed when 1 mMofaptamers were incubated with various number of MCF7 cells. Theconcentration of aptamer greater than 1 mM did not show anysignificant changes. Therefore, the optimum concentration ofaptamer was selected to be 1 mM for our subsequent experiments.
3.4. Sensitivity of experiment
The sensivity of this colorimetric aptasensor was determined
Fig. 2. Absorption spectra (A) and colorimetric changes (B) after incubation with different numbers of cells (aee, 105, 104, 103, 102, 10 MCF-7 cells, respectively) and (C) The linearcalibration curve of A660/A520 ratio MCF-7 cells.
Fig. 3. Evaluation of aptamer interaction with two other cell lines at different numbers(105,104,103,102,10 cells/ml). Cells were incubated with the aptamer for 1 h aftercentrifugation, supernatant was studied using complementary probe1/2-AuNPs.
using different numbers of MCF7 cells (0e105 cells/ml). The result(Fig. 2) shows that the solution, color changed from red to purplewhen we increased the concentration of MCF-7 cells. This phe-nomenon was due to the aptamers uptake by MCF-7 cells. Plottingthe absorption ratio versus the number of cells (10e105), a linearslope was observed with a regression coefficient of 0.996. Thedetection limit was determined to be 10 cells for MCF-7 cells usingaptasensor which is comparable to previous reported number asseen in Table 1. Therefore, our proposed aptasensor methoddemonstrated to be highly sensitive, accurate and less laboriousthat enable us to detect cancer cells (MCF-7) [25e30]. Recently,colorimetric detection of cancer cell based on dual e aptamer hasbeen also reported by E. Wang et al. [30].
3.5. Selectivity of experiment
The selectivity of this colorimetric aptasensor was evaluated fortwo other types of cancer, human lung cancer cell line (A549) andhuman gastric cancer cell line (AGS) as negative cells. As it is shownin Fig. 3, when the negative cells were incubated with the aptamerat the same concentration of MCF-7, negligible changes in absor-bance intensities were observed in comparison with MCF-7 cells.They showed slight decrease independent from the number of cells,which might be due to their different nucleolin over-expression. Itwas concluded that the specific nucleolin receptors on the surface
Table 1Comparison of different methods for detection of MCF-7 cells.
Method Linear range/cells/ml Detection limit/cells/ml Reference
Electrochemiluminescent detection 500 � 2 � 107 230 [25]cyclic voltammetry detection 1 � 105 � 1 � 108 1 � 105 [26]Photoluminescence (PL) and square-wave voltammetric (SWV) assays 250e104 201 and 85 [27]Electrochemiluminescent detection based on the resonance energy transfer 100e2500 30 [28]Fluorescence resonance energy transfer 50e3000 36 [29]Colorimetric detection via dual eaptamer target binding strategy 10e105 10 [30]Colorimetric aptasensor based on aggregation of gold nanoparticles 10e105 10 This work
of A549 and AGS cells may prohibited the uptake of aptamers andresulted to different absorbance pattern. As it was expected, also nochanges were observed in the absorbance intensity for primaryfibroblast cells. The obtained results were in close agreement withthe fact that no aptamer uptake occurs in the absence of nucleolinreceptors on the cell membrane. These results clearly indicate thatthe nanobiosensor can efficiently distinguish the cancer cells fromnormal cells with excellent selectivity through aptamer bindingcapability and specificity.
4. Conclusion
In conclusion, we have demonstrated a colorimetric method forhighly efficient detection of rare circulating cancer cells. Thismethod could provide a detection tool for breast cancer cells as wellas other types of human cancers with high sensitivity and highselectivity. The detection limit of this proposed method was esti-mated about 10 cells. The improvement in analytical properties islikely due to the good efficiency and specificity of hybridization,verified by UVevisible spectroscopy and TEM imaging analysis. Thismethod had significant advantages including high sensitivity,specificity and simple operation. In contrast to expensive fluores-cence and electrochemical based assays, this colorimetric methoduses UVevis spectroscopy and makes it possible to detect cancercells by naked eye. It is also a separation free method for cellanalysis that avoids complex operations, such as cell-immobilization and washing procedures. The study also showedthe novel exploitation of specific features of cancer cells that is cellsurface receptors for efficient and specific detection of them. Themethod is generic and can be applied for other cancer cell analysesand clinical diagnosis as well.
Acknowledgments
The authors are grateful to the Research Council of University ofTehran (Grant 28645/01/01) for the financial support of this work.
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