aptamer-conjugated nanoparticles for selective collection and detection of cancer cells

Aptamer-Conjugated Nanoparticles for SelectiveCollection and Detection of Cancer Cells

Joshua K. Herr, Joshua E. Smith, Colin D. Medley, Dihua Shangguan, and Weihong Tan*

Center for Research at the Bio/Nano Interface, Department of Chemistry and Shands Cancer Center, UF Genetics Instituteand McKnight Brain Institute, University of Florida, Gainesville, Florida 32611

We have developed a method for the rapid collection anddetection of leukemia cells using a novel two-nanoparticleassay with aptamers as the molecular recognition element.An aptamer sequence was selected using a cell-basedSELEX strategy in our laboratory for CCRF-CEM acuteleukemia cells that, when applied in this method, allowsfor specific recognition of the cells from complex mixturesincluding whole blood samples. Aptamer-modified mag-netic nanoparticles were used for target cell extraction,while aptamer-modified fluorescent nanoparticles weresimultaneously added for sensitive cell detection. Com-bining two types of nanoparticles allows for rapid, selec-tive, and sensitive detection not possible by using eitherparticle alone. Fluorescent nanoparticles amplify thesignal intensity corresponding to a single aptamer bindingevent, resulting in improved sensitivity over methodsusing individual dye-labeled probes. In addition, aptamer-modified magnetic nanoparticles allow for rapid extractionof target cells not possible with other separation methods.Fluorescent imaging and flow cytometry were used forcellular detection to demonstrate the potential applicationof this method for medical diagnostics.

Accurate, sensitive methods for leukemia diagnosis facilitatethe selection of effective therapeutic pathways by clinicians. Assaysfor sensitive minimal residual disease detection are also essentialfor monitoring disease development and distinguishing those whoare more susceptible to relapse. Current methods for leukemiadiagnosis apply combinations of bone marrow and peripheralblood cytochemical analyses including karyotyping,1 immunophe-notyping by flow cytometry2 or microarray,3 and amplification ofmalignant cell mutations by PCR.4

Immunophenotypic analyses of leukemia cells use antibodyprobes to exploit the variation of specific surface antigens in orderto differentiate malignant cells from normal cell lines. Thelimitation to this method is that antigens used for cell recognitionare normally not exclusively expressed on any single cell type,

dramatically influencing sensitivity, and resulting in false positivesignals. Because of this, immunophenotypic analyses often requiremultiple antibody probes for accurate cell detection, increasingboth the complexity and the cost of the method.

PCR-based methods have proven to be highly sensitivediagnostic techniques for cellular recognition,4-6 but they areindirectly detecting cells by monitoring RNA expression andrequire prolonged RNA isolation steps before analysis. In addition,the variable sensitivity of PCR can limit its effectiveness as adiagnostic technique and can lead to false-negative results,particularly with occult tumor cells where low-level signals areexpected.4 Immunophenotypic analyses are also time-consumingand costly, and therefore, there is still a need to develop newtechnologies for rapid, economical cell recognition.

Here an assay using aptamer-conjugated nanoparticles isdescribed for the rapid detection of acute leukemia cells usinghigh-affinity DNA aptamers for signal recognition. An 88-baseoligonucleotide sequence with specific binding properties (Kd )5 nM) for CCRF-CEM acute leukemia cells was attached tomagnetic and fluorescent nanoparticles in order to develop aspecific platform for collecting and imaging intact target leukemiacells from mixed cell and whole blood samples.

Highly specific DNA aptamers are selected by SELEX7,8 to bindwith specific molecular or cellular targets. Of late, aptamers havebeen recognized as reliable affinity ligands, which rival antibodiesin their diagnostic potential.9 While antibodies are still extractedand purified from animals, aptamers can be easily synthesizedfor the analysis of molecules unlimited by toxicity and withoutanimal destruction.10 Aptamers11-15 are able to fold into unique

* To whom correspondence should be addressed. E-mail: [email protected]: 352-846-2410. Fax: 352-846-2410.(1) Faderl, S.; Kantarjian, H. M.; Talpaz, M.; Estrov, Z. Blood 1998, 91, 3995-

4019.(2) Paredes-Aguilera, R.; et al. Am. J. Hematol. 2001, 68, 69-74.(3) Belov, L.; de la Vega, O.; dos Remedios, C. G.; Mulligan, S. P.; Christo-

pherson, R. I. Cancer Res. 2001, 61, 4483-4489.(4) Ghossein, R. A.; Bhattacharya, S. Eur. J. Cancer 2000, 36. 1681-1694.

(5) Iinuma, H.; Okimaga, K.; Adachi, M.; Suda, K.; Sekine, T.; Sakagawa, K.;Baba, Y.; Tamura, J.; Kumagai, H.; Ida, A. Int. J. Cancer 2000, 89, 337-344.

(6) Liu Yin, J. A.; Grimwade, D. Lancet 2002, 360, 160-162.(7) Ellington, A. D.; Szostak, J. W. Nature 1990, 346, 816-820.(8) Tuerk, C.; Gold, L. Science 1990, 249, 505-510.(9) Brody, E. N.; Gold, L. Rev. Mol. Biotechnol. 2000, 74, 5-13.

(10) Tombelli, S.; Minunni, M.; Mascini, M. Biosens. Bioelectron. 2005, 20,2424-2434. German, I.; Buchanan, D. D.; Kennedy R. T. Anal. Chem. 1998,70, 4540-4545.

(11) Kirby, R.; Cho, E. J.; Gehrke, B.; Bayer, T.; Park, Y. S.; Neikirk, D. P.;McDevitt, J. T.; Ellington, A. D. Anal. Chem. 2004, 76, 4066-4075. Tan,W.; Wang, K.; Drake, T. Curr. Opin. Chem. Biol. 2004, 8 (5), 547-553.

(12) Yang, C. J.; Jockusch, S.; Vicens, M.; Turro, T.; Tan, W. Proc. Natl. Acad.Sci. U.S.A. 2005, 102, 17278-17283. Li, J.; Fang, X.; Tan, W. Biochem.Biophys. Res. Commun. 2002, 292 (1), 31-40.

(13) Osborne, S. E.; Matsumura, I.; Ellington, A. D. Curr. Opin. Chem. Biol.1997, 1, 5-9.

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Anal. Chem. 2006, 78, 2918-2924

2918 Analytical Chemistry, Vol. 78, No. 9, May 1, 2006 10.1021/ac052015r CCC: $33.50 © 2006 American Chemical SocietyPublished on Web 03/24/2006

three-dimensional conformations with distinct biomolecular bind-ing properties and have successfully been used for proteindetection by sensor array and affinity capillary electrophoresis,and for targeted therapeutic applications, including a biodegrad-able nanoparticle-aptamer-based method for targeted drug de-livery to specific prostate cancer cells and many other interestingapplications.

Tumor cell SELEXsan in vitro process identifying DNAsequences with strong affinities toward intact tumor cellsswasused to select an aptamer with high specificity toward our targetleukemia cell line. Aptamers selected by cell SELEX have theability to differentiate between numerous types of cells. Thesenatural discriminatory properties are revealed during the selectionprocess. Following the published protocols, we have selected anaptamer for acute leukemia cells with a sequence of TTTAAAA-TACCAGCTTATTCAATTAGTCACACTTAGAGTTCTAGCTG-CTGCGCCGCCGGGAAAATACTGTACGGATAGATAGTAAGTGCAATCT-3′.We hypothesized that combining the selective characteristics ofaptamers with magnetic nanoparticle-based separation couldproduce a universal, selective, and sensitive method for thecollection and subsequent detection of various target molecules.Our aptamer was attached to fluorescent nanoparticles to provideenhanced signal and a means of detection. Beneficial aspects ofstable luminescent probessspecifically high sensitivity and easeof detectionsfacilitate biological and nanoscale imaging analyses.Dye-doped silica nanoparticles have previously been used toreplace fluorescent dyes because of their signal amplification andcompatibility for the immobilization of biomolecules.16-18 Exploit-ing the availability of hydroxyl groups on the particle surface hasproven useful for DNA and mRNA detection,17,19 as well as proteinand antigen detection.19-24 Here we have utilized fluorescentnanoparticles to enhance the signal intensity corresponding toeach aptamer binding event. For each fluorescent nanoparticlebound to a target cell via aptamer, a silica nanoparticle containingthousands of dye molecules is immobilized on the cell surface.Upon excitation, those dye molecules simultaneously release afluorescent signal that is significantly brighter than an individualdye probe.

As an alternative to centrifugation, magnetic nanoparticle-aptamer-based cell sorting was employed here for selectivemalignant cell collection. Previously, magnetic activated cellsorting (MACS) was used extensively for selective extraction and

enrichment of epithelial cells,25 endothelial cells,26 bacteria,27 andcirculating tumor cells.5,28-30 While these methods normally usemicrometer-sized magnetic polymer beads, we have chosen toutilize 65-nm silica-coated magnetic nanoparticles. Magnetic nano-particles have previously been used for gene collection31 andpeptide isolation for MS analysis.32 The small size and increasedrelative surface area of nanoparticles provide enhanced extractioncapabilities compared with larger particles.31 We employed aMACS technique using aptamer-modified iron oxide-doped nano-particles for selective leukemia cell extraction. In addition toenabling selective target extraction, magnetic nanoparticle-basedsorting removes the need to centrifuge cell samples and the needfor presample cleanup. As a result, the collection of unwantednanoparticle aggregates and unbound materials from target cellextractions is eliminated, and a reduced background is observed.

Fluorescence imaging and flow cytometry were used to confirmthe selectivity and enhanced sensitivity of the assay. The simul-taneous use of two aptamer-modified nanoparticles for targetedcell collection and detection allows for rapid, accurate analysis oftarget cells not possible by either particle alone. This method alsodemonstrates the capacity to reproducibly extract target cells fromcomplex mixtures and whole blood samples, establishing afoundation for the relevance of this method for clinical applications.

METHODS AND MATERIALSMaterials. All materials were purchased from Sigma-Aldrich

(St. Louis, MO) unless other noted. Whole blood samples wereobtained from Research Blood Components, LLC (Brighton, MA).Fluo-4 was purchased from Molecular Probes (Eugene, OR), andcarboxylethylsilanetriol sodium salt was purchased from Gelect,Inc. (Morrisville, PA). N-Hydroxysulfosuccinimide (Sulfo-NHS)and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride(EDC) were purchased from Pierce Biotechnology, Inc. (Rockford,IL). Hydrochloric acid and ammonium hydroxide were obtainedfrom Fisher Scientific.

Fluorescent Nanoparticle Synthesis. Dye-doped nanopar-ticles were synthesized by the reverse microemulsion method.21

First, 1.77 mL of Triton X-100, 7.5 mL of cyclohexane, and 1.6 mLof 1-hexanol were added to a 20-mL glass vial with constantmagnetic stirring. Then, 400 µL of H2O and 80 µL of 0.1 M tris-(2,2′ -bipyridyl)dichlororuthenium(II) hexahydrate (Rubpy) dye(MW ) 748.63) were added, followed by the addition of 100 µLof tetraethyl orthosilicate (TEOS). After 30 min of stirring, 60 µLof NH4OH was added to initiate silica polymerization. After 18 h,the carboxyl-modified silica postcoating was initiated by adding50 µL of TEOS, 40 µL of carboxylethylsilanetriol sodium salt, and

(15) Daniels, D. A.; Chen, H.; Hicke, B. J.; Swiderek, K. M.; Gold, L. Proc. Natl.Acad. Sci. U.S.A. 2003, 26, 15416-15421. Fang, X.; Sen, A.; Vicens, M.;Tan, W. ChemBioChem 2003, 4, 829-834.

(16) X. Zhao, L.; Hilliard, S.; Mechery, Y.; Wang, R.; Bagwe, S.; Jin, W.; Tan, W.Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 15027-32.

(17) Zhao, X.; Tapec-Dytioco, R.; Tan, W. J. Am. Chem. Soc. 2003, 125, 11474-11475.

(18) Zhao, X.; Bagwe, R.; Tan, W. Adv. Mater. 2004, 16, 173.(19) Lian, W.; Litherland, S.; Badrane, H.; Tan, W.; Wu, D.; Baker, H.; Gulig, P.;

Lim, D.; Jin, S. Anal. Biochem: 2004, 334, 135-144.(20) Yang, W.; Zhang, C. G.; Qu, H. Y.; Yang, H. H.; Xu, J. G. Anal. Chim. Acta

2004, 503, 163-169.(21) Santra, S.; Wang, K.; Tapec-Dytioco, R.; Tan, W. J. Biomed. Opt. 2001, 6.(22) Santra, S.; Zhang, P.; Wang, K.; Tapec, R.; Tan, W. Anal. Chem. 2001, 73,

4988-4993. Wang, L.; Wang. K. M.; Santra, S.; Zhao, X. J.; Hilliard, L. R.;Smith, J. E.; Wu, J. R.; Tan, W. H. Anal. Chem. 2006, 78, 6469654.

(23) Yang, H.; Qu, H.; Lin, P.; Li, S.; Ding, M.; Xu, J. Analyst 2003, 128, 462-466.

(24) Ye, Z.; Tan, M,; Wang, G.; Yuan, J. Anal. Chem. 2004, 76, 513-518.

(25) Griwatz, C.; Brandt, B.; Assmann, G.; Zanker, K. S. J. Immun. Methods 1995,183, 251-265.

(26) Marelli-Berg, F. M.; Peek, E.; Lidington, E. A.; Stauss, H. J.; Lechler, R. I.J. Immun. Methods 2000, 244, 205-215.

(27) Porter, J.; Robinson, J.; Pickup, R.; Edwards, C. J. Appl. Microbiol. 1998,84, 722-732.

(28) Stanciu, L. A.; Shute, J.; Holgate, S. T.; Djukanovic, R. J. Immun. Methods1996, 189, 107-115.

(29) Hu, X. C.; Wang, Y.; Shi, D. R.; Loo, T. Y.; Chow, L. W. C. Oncology 2003,64, 160-165.

(30) Benez, A.; Geiselhart, A.; Handgretinger, R.; Schiebel, U.; Fierlbeck, G. J.Clin. Lab. Anal. 1999, 13, 229-233.

(31) Zhao, X.; Tapec-Dytioco, R.; Wang, K.; Tan, W. Anal. Chem. 2003, 75,3476-3483.

(32) Turney, K.; Drake, T. J.; Smith, J. E.; Tan, W.; Harrison, W. W. Rapid Comm.Mass Spectrom. 2004, 18, 1-8.

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10 µL of 3-(trihydroxyl)propyl methyl phosphonate. Polymerizationproceeded for 18 h, and particles were centrifuged, sonicated, andvortexed four times with 95% ethanol, followed by one wash withH2O. Carboxyl-functionalized Rubpy nanoparticles were modifiedwith DNA by adding 1.2 mg of EDC, 3.5 mg of Sulfo-NHS, and0.5 nmol of DNA with 2 mg of particles dispersed in 1.5 mL of 10mM MES buffer (pH 5.5). The solution was then mixed for 3 h.Particles were washed by centrifugation at 14 000 rpm three timeswith 0.1 M phosphate-buffered saline (PBS) (pH 7.2). Rubpynanoparticles were stored at room temperature and were dis-persed in cell media buffer at a final concentration of ∼10 mg/mL.

Magnetic Nanoparticle Synthesis. The iron oxide coremagnetic nanoparticles32 were prepared by means of precipitatingiron oxide by mixing ammonia hydroxide (2.5%) and iron chlorideat 350 rpm using a mechanical stirrer (10 min). The iron chloridesolution contains ferric chloride hexahydrate (0.5 M), ferrouschloride tetrahydrate (0.25 M), and HCl (0.33 M). After threewashes with water and once with ethanol, an ethanol solutioncontaining ∼1.2% ammonium hydroxide was added to the ironoxide nanoparticles, yielding a final concentration of ∼7.5 mg/mL.

To create the silica coating for the magnetite core particles,tetraethoxyorthosilicate (200 µL) was added, and the mixture wassonicated for 90 min to complete the hydrolysis process. Forpostcoating, an additional aliquot of TEOS (10 µL) was added andadditional sonication was performed for 90 min. The resultingnanoparticles were washed three times with ethanol to removeexcess reactants.

For avidin coating, a 0.1 mg/mL Fe3O4-SiO2 (silica-coatedmagnetic nanoparticles) solution and a 5 mg/mL avidin solutionwere mixed and then sonicated for 5-10 min. The mixture wasincubated at 4° C for 12-14 h. The particles were then washedthree times with 10 mM PBS, pH 7.4, and dispersed at 1.2 mg/mL in 100 mM PBS, and the avidin coating was stabilized by cross-linking the coated nanoparticles with 1% glutaraldehyde (1 h at25° C). After another separation, the particles were washed threetimes with 1 M Tris-HCl buffer. Then, the particles were dispersedand incubated in the 1 M Tris-HCl buffer (3 h at 4 °C), followedby three washes in 20 mM Tris-HCl/5 mM MgCl2, pH 8.0.

DNA was attached to the particles by dispersing the particlesat 0.2 mg/mL in 20 mM Tris-HCl, 5 mM MgCl2, pH 8.0. Biotin-labeled DNA was added to the solution at a concentration of 31µM. The reaction was incubated at 4 °C for 12 h, and three finalwashes of the particles were performed using 20 mM Tris-HCl, 5mM MgCl2, at pH 8.0. Magnetic nanoparticles were used at a finalconcentration of ∼0.2 mg/mL and stored at 4 °C before use.

Magnetic Extraction. For each magnetic extraction, thespecified amount of magnetic nanoparticles was added to thesample. The aptamer-conjugated magnetic nanoparticles were thenincubated with the target cells for 5 min unless specifiedotherwise. After the incubation period a magnetic field was appliedto the side of the sample container. After 1 min, the nonmagneticmaterials were removed with a Pasteur pipet, fresh buffer wasadded, and the magnetic field was removed. The materials weremixed in the buffer, and previous steps were repeated for a totalof three times to remove anything nonspecifically bound to themagnetic nanoparticles.

Cells. CCRF-CEM cells (CCL-119 T-cell, human acute lym-phoblastic leukemia) and Ramos cells (CRL-1596, B-cell, humanBurkitt’s lymphoma) were obtained from the American TypeCulture Association and cultured in RPMI 1640 medium supple-mented with 10% fetal bovine serum and 100 IU/mL penicillin-streptomycin. Before nanoparticle incubation, cells were dispersedin 500 µL of cell media buffer and centrifuged at 920 rpm for 5min three times and were then redispersed in 200 µL of mediabuffer. Fluorescent and magnetic nanoparticle solutions were thensimultaneously added to the cell solutions at a 20:1 ratio,respectively. After nanoparticle incubation, cells were washed bymagnetic extraction with 500 µL of media buffer three times andredispersed in 20 µL of buffer for imaging and 200 µL of bufferfor flow cytometric and collection efficiency analyses. All puresample experiments started with 1.0 × 105-5.0 × 105 cells beforenanoparticle incubation.

Sample Assays. To determine the extraction and detectioncapabilities in an artificial complex sample, equal amounts of CEMand Ramos cells were mixed and tested using the assay. Ap-proximately 105 cells of each type were mixed, followed bymagnetic and fluorescent nanoparticle incubation for 5 min.Magnetic extraction procedures were performed three times toremove unbound cells. A 2-µL aliquot of the redispersed extractedsample was then imaged by confocal microscopy.

To show applicability in real biological samples, whole bloodwas spiked with 105 CEM cells. Fluorescent and magneticnanoparticles were then incubated for 5 min with spiked andunspiked blood samples, followed by three magnetic extractions.Confocal imaging was then used to characterize cell extractions.

Collection efficiency was measured from pure cell samples andspiked blood samples. For efficiency studies, cell samples sub-jected to nanoparticle incubation and magnetic extractions werecompared to samples not subjected to any separations by magneticextraction. For pure cell analyses, 5-30 µg of magnetic nanopar-ticles were individually incubated in 5-µg increments with ∼105

cells initially and subjected to magnetic extractions after 5-minincubation. The efficiency of cell extraction from the spiked bloodsample was determined by incubating magnetic nanoparticles (30µg) with 500 µL of whole blood spiked with 105 CEM cells. Cellswere counted by flow cytometry for pure samples and by imagingfor blood samples. Various magnetic nanoparticle concentrationswere used to determine maximum collection efficiency and optimalseparation efficiency.

Cell Imaging. Fluorescence imaging was conducted with aconfocal microscope setup consisting of an Olympus IX-81 invertedmicroscope with an Olympus Fluoview 500 confocal scanningsystem and three lasers, a tunable argon ion laser (458, 488, 514nm), a green HeNe laser (543 nm), and a red HeNe laser (633nm) with three separate photomultiplier tubes for detection. Thecellular images were taken with a 20 × 0.70 NA objective. Thefluorescent nanoparticles were excited with the 488-nm line of theargon ion laser, and emission was detected using a 610-nm long-pass filter. Fluo-4 was excited with the 488-nm laser line and wasdetected with a 505-525-nm band-pass filter.

Flow Cytometry. Fluorescence measurements were also madeusing a FACScan cytometer (Becton Dickinson ImmunocytometrySystems, San Jose, CA). To support imaging data, Rubpy fluores-cence of pure samples initially containing 105 cells were measured

2920 Analytical Chemistry, Vol. 78, No. 9, May 1, 2006

by counting 30 000 events. Cell experiments were performedexactly as stated for imaging experiments, except all solutionswere diluted to a final volume of 200 µL. Cell sorting allowed foraccurate quantitative analysis of cell samples, as well as a platformfor collection efficiency determination.

RESULTS AND DISCUSSIONCollection Efficiency. Values for the collection efficiency were

obtained by incubating increasing amounts of magnetic nanopar-ticles with the target CEM cells and Ramos control cells. Thenumber of cells collected was determined by flow cytometry bythe counting of signal events. In addition, the cell counting wasperformed on a control sample of both cell types that did notundergo the magnetic extraction and was taken as the totalamount of the cells. The collection efficiency was calculated bydividing the number of events for each sample by the total cellnumber. As seen in Figure 1, the collection efficiency of targetcells from ranges from 30 to 80%; however, the collection efficiencyseems to plateau at ∼80%. In addition, the Ramos control cellshad collection efficiencies ranging from 0.5 to 5% for the same

magnetic nanoparticle concentrations. This indicates that thetarget cells can be preferentially extracted from a sample, whilefew of the Ramos cells are extracted using the same method. Sincethe use of 10 µL of magnetic nanoparticles had a high separationefficiency, this amount was used for sample assay experimentsto allow for binding of both nanoparticle types to the same cell.

Dye and Nanoparticle Fluorescent Intensity Comparison.To demonstrate the fluorescence enhancement capabilities ofRubpy-doped nanoparticles, individual Rubpy probes were linkedwith our DNA aptamer and directly compared to Rubpy nanopar-ticle-aptamer conjugates after immobilization on our target cells.Equal concentrations of magnetic and Rubpy nanoparticles (0.5nM) were incubated with CEM cells, then washed by magneticextraction with 500 µL of media buffer three times, and redis-persed in 20 µL of buffer for imaging and 200 µL of buffer forflow cytometric analysis. Panels A and B in Figure 2 compare cellextractions labeled with fluorescent nanoparticles to extractionslabeled with Rubpy dye. There is a significant difference in theamount of fluorescent signal seen in the two images. Flowcytometry was used to verify that the Rubpy nanoparticles provideenhanced fluorescence signal, and Figure 2C confirms over a 100-fold enhancement of Rubpy nanoparticle-labeled cells to Rubpydye-labeled cells. This figure also shows the nanoparticle-labeledcells in an apparent bimodal distribution. While the exact causeof this pattern is unknown, possible explanations include theformation of nanoparticle aggregates, formation of cell/nanopar-ticle aggregates, different levels of receptors on cells, or simplyan artifact of the experimental method used. Nonetheless, theexperiment illustrates the signal advantage that the fluorescentnanoparticles possess over single fluorophores.

Sample Assays. To demonstrate the concept of our twoparticle-based magnetic collection and detection technique, indi-vidual CEM and Ramos cell solutions were subjected to our two-

Figure 1. Flow cytometric determination of magnetic nanoparticlecollection and separation efficiencies between target and control cells.

Figure 2. Fluorescence images of extracted samples after 5min incubation with (A) 40 µM Rubpy dye-aptamer conjugates and (B) 0.5 nMRubpy nanoparticle-aptamer conjugates, followed by three magnetic washes. (C) Comparison of dye-labeled cells to nanoparticle-labeled cellsby flow cytometric analysis.


particle procedures, followed by fluorescent imaging and flowcytometric analysis. Before nanoparticle incubation, cells weredispersed in 500 µL of cell media buffer, centrifuged three timesat 920 rpm for 5 min, and then redispersed in 200 µL of mediabuffer. Fluorescent and magnetic nanoparticle solutions were thensimultaneously added to the cell solutions at a 20:1 ratio,respectively. After 5-min nanoparticle incubation, cells werewashed by magnetic extraction with 500 µL of media buffer threetimes and redispersed in 20 µL of buffer for imaging and 200 ofµL buffer for flow cytometric analyses. All pure sample experi-ments started with 1.0 × 105-5.0 × 105 cells before nanoparticleincubation. Each pure cell extraction was repeated 10 times.

Figure 3 shows representative confocal images of 2-µL aliquotsof target cells (A) and control cells (B) after 5-min incubation andthree magnetic extractions.

There is a noticeable change in both the amount of cellspresent and fluorescent signal between the extracted cell solutions.Magnetic collection pulled out few control cells, while a significantnumber of target cells were extracted using the same procedures.In addition, the few control cells inadvertently collected bymagnetic extractions were labeled with few Rubpy nanoparticlesand had no significant fluorescent signal. Conversely, the targetCEM cells that were subjected to the assay had very intensefluorescent signals that made them easily distinguishable from

Figure 3. Images of extracted samples from (A) target cells and (b) control cells and (C) flow cytometric comparison of target and controlsignal after 5-min incubation with magnetic and fluorescent nanoparticles, followed by three washes by magnetic separation.

Figure 4. Images of (A) 1:1 ratio of target cells mixed with Fluo-4-stained control cells. (B) Fluo-4 signal and (C) Rubpy signal after 5 min,two-particle incubation and three magnetic washes of the mixture in Figure 3A. (D) 1:1 ratio of Fluo-4-stained target cells mixed with controlcells. (E) Fluo-4 signal and (F) Rubpy signal after 5 min, two-particle incubation and three magnetic washes of the mixture in Figure 3D.


the control cells. The flow cytometric analysis of the pure sampleassay, Figure 3C confirms that fewer control cells were collectedthan target cells, and the control cells showed less fluorescentemissions than the extracted target cells.

Mixed Cell Sample Assays. To evaluate the potential of theassay, complex samples needed to be tested to determineextraction and detection capabilities in complex matrixes. InFigure 4 we show the results from our artificial complex samplewhere equal amounts of CEM and Ramos cells were mixed andour two-particle assay was applied. To differentiate CEM fromRamos cells, Fluo-4sa fluorescent calcium indicatorswas usedto label Ramos cells prior to nanoparticle incubation. Fluo-4-labeledcontrol cells were mixed (1:1) with unlabeled CEM cells shownin Figure 4A. Magnetic and fluorescent nanoparticles weresimultaneously added and incubated at 4 °C for 5 min withoccasional gentle stirring. After incubation, a magnetic field wasapplied to remove cells that were not attached to the aptamer-labeled iron oxide particles. A 2-µL aliquot of the extracted samplewas then illuminated to monitor Fluo-4 and Rubpy fluorescenceas shown in Figure 4B and C, respectively. Based on the images,the assay was able to collect the CEM cells in the sample andbright fluorescence from the Rubpy nanoparticles made themeasily distinguishable. The experiment was also performed bylabeling CEM cells with Fluo-4 and mixing them with unlabeledcontrol cells as in Figure 4D. The cells shown in Figure 4E wereseparated by the two-particle assay, and all exhibit a Fluo-4 signal.In Figure 4F, the same cells are shown with the Rubpy emissionoverlaid. The presence of the Fluo-4 fluorescence proves that onlythe CEM cells were collected and imaged. The lack of Fluo-4

signal in Figure 4B, along with the presence of the Fluo-4 signalin Figure 4E, proves that only target cells are being collected usingthis method for extractions from 1:1 cell mixtures. These sampleassays were repeated five times with similar results achieved foreach experiment.

Whole Blood Sample Assays. Blood samples were also usedto determine detection capabilities from complex biologicalsolutions. Control experiments indicated that the aptamer se-quence used was stable in serum samples for up to 2 h. Targetcells were spiked into whole blood samples (500 µL) andcompared to unspiked samples after magnetic extraction to makecertain that target cells could be detected in complex biologicalsamples. As shown in Figure 5, nonspecific interactions causedthe unwanted collection of some red blood cells, but the lack ofRubpy fluorescent signal on the unwanted cells allows for targetcells to still be accurately distinguished. For magnetic extractionsfrom whole blood samples, 40% of the spiked target cells wereroutinely recovered after three magnetic washes and after ac-counting for dilution. This is consistent with current extractionefficiency values reported by immunomagnetic separation.28,30

These experiments were repeated for total of five times withsimilar results being obtained in each sample.

This experiment was meant to mimic a real clinical sample,which normally would contain thousands of different species. Bysuccessfully extracting our target cell line from whole blood, wehave shown that this method is applicable for biomolecular andcellular detection in real clinical applications. Our assay selectivelyremoves our target cells from this complex mixture with collectionefficiencies rivaling or surpassing current methods for cellular

Figure 5. Confocal images of extractions from whole blood. (A) Extracted sample from target cell spiked whole blood. (B) Extraction fromunspiked whole blood. (C) and (D) show magnified images of extracted cells from Figure 4A.


detection from clinical samples.Discussion. The utilization of aptamer-conjugated magnetic

and fluorescent nanoparticles in this assay was possible onlybecause there are sufficient aptamer binding sites for both typesof particles on our target leukemia cells. For the analysis of cellshaving few aptamer recognition sites, different aptamers or otherrecognition elements can be labeled on each type of particle toeliminate competitive binding. Compared with current diagnostictechniques, the two-particle assay described has three distinctadvantages for molecular recognition. First, highly specific aptam-ers were used for molecular recognition. Prolonged stability andfacile synthesis make aptamers an ideal replacement for antibodiesin cellular recognition studies. We have shown that incorporatingaptamers onto nanoparticles does not adversely affect their bindingproperties with intact cells, and therefore, they can be utilizedfor selective extraction and sensitive molecular detection. Theaptamer used was selected specifically for intact target cells, andcellular detection is possible without significant sample prepara-tion.

The second major advantage of the two-particle assay ismagnetic nanoparticle-aptamer-based cell sorting, which allowsfor selective cell collection from complex samples. Iron oxide-doped silica nanoparticle-aptamer conjugates were used here fort-cell collection and washing. Our aptamer-based MACS applicationis effective for the selective extraction of target molecules andallows for enhanced extraction efficiency from clinical samples.Another advantage of magnetic extraction is the removal ofnanoparticle aggregates and other unbound fluorescent materialsthat normally would cause increased background fluorescence.

The two-particle assay is also very fast, with rapid incubationand magnetic extractions allowing for rapid detection. Whileimmunophenotypic and PCR-based analyses take hours to com-plete, our two-particle assay requires as little as 5-min incubationfor sufficient nanoparticle binding, and the entire method caneasily be performed in less than 1 h.

Fluorescent dye-doped nanoparticles were used to provideenhanced signaling capabilities. Rubpy-doped nanoparticle-aptamer conjugates were used to amplify the signal intensitycorresponding to each aptamer binding event, resulting in much

improved sensitivity compared to individual Rubpy dye-labeledprobes. Nanoparticles were coated with silica, and dye concentra-tions inside the nanoparticles were optimized to reduce photo-bleaching effects, further enhancing the method’s sensitivity.Rubpy nanoparticles are shown here to increase the fluorescentsignal corresponding to aptamer binding to our target leukemiacells and have been used here as effective, sensitive replacementsfor individual Rubpy-labeled aptamer probes. The fluorescentnanoparticles also add an additional level of selectivity to themethod since only cells that are magnetically extracted andpossess a high fluorescent intensity are recognized as target cells.As in the whole blood experiments, some cells were nonspecifi-cally extracted but were easily distinguished from the target cellsbased on the fluorescence intensity.

Though Rubpy nanoparticles have shown an enhancementhere over individual dye-labeled probes, the true benefit of thisassay will be revealed when the enhancement effects of nanopar-ticles are used to detect binding with low expression cell surfacemarkers. This is crucial since there are many markers that arefew in number on the cell surface that cannot be detected usingcurrent dye-labeling methods. Further optimization of this methodmay prove necessary for applications in clinical diagnostics;nevertheless, this proof of concept has demonstrated the potentialapplicability of this method for rapid cellular and moleculardetection. The enhanced selectivity of our assay has shownpromise for extraction and detection of target cells from wholeblood, and additional investigation may prove this method valuablefor the detection of numerous molecular analytes from blood orother complex biological mixtures.

ACKNOWLEDGMENTThe authors acknowledge Ms. Hui Lin for her expertise in

DNA synthesis. This work was partially supported by a NSF NIRTgrant and NIH grants.

Received for review November 13, 2005. AcceptedJanuary 19, 2006.

AC052015R


aptamer-conjugated nanoparticles for selective collection and detection of cancer cells

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