BioChip J. (2010) 4(1): 42-48

DOI 10.1007/s13206-010-4107-y

Abstract Here we present telomerase activity screen-ing using a capillary electrophoretic (CE) microdevicefor human cancer diagnostics. The telomerase enzyme,a contributor to the maintenance of telomere length incancer cells, was extracted from various human cellsincluding MCF-7 (Human breast cancer cells), A549(Human lung cancer cells), and SK-N-SH (Humanneuroblastoma cells), and then telomeric repeat ampli-fication protocol (TRAP)-based genetic analysis wasperformed to evaluate the activity of telomeraseenzyme. The resultant 6-bp tandem repeat PCR ampli-cons derived from cancer cells were separated anddetected on the 6 cm-long CE microdevice within 5min. A limit of detection of 5 cells was achieved toanalyze the telomerase activity on a CE microchip. Incomparison with a conventional PAGE method, micro-fabricated CE-based analysis has many advantages interms of high speed, low sample consumption, andhigh sensitive detection. The successful demonstra-tion of telomerase activity screening on a chip impliesgreat potential of a complete integration of a samplepreparation and PCR unit in a single format for high-performance cancer diagnostics in a clinical arena.

Keywords: Telomerase, Telomeric repeat amplifica-tion, Polymerase chain reaction, Capillary electropho-retic microdevice, Cancer diagnostics

Introduction

Telomeres are specific structures found at the end of

eukaryotic chromosomes. Telomeric DNA is charac-terized by an array of tandemly repeated, G-rich DNAsequences that are highly conserved during evolutionand consists of short repeated sequences of the hexa-nucleotide 5′-TTAGGG-3′1. Telomerase is a ribonu-cleoprotein to catalyze the addition of TTAGGGrepeats to the ends of vertebrate chromosomes, usinga complementary sequence of its intrinsic RNA com-ponent as a template2,3. Telomerase activity has beenshown to be specifically expressed in immortal cells,cancer and germ cells4,5 where it compensates for telo-mere shortening during DNA replication and thus sta-bilizes telomere length6,7. Because of its involvementin carcinogenesis, telomerase activity has been knownas a promising biomarker and prognostic indicator ofcancer, and an attractive target for chemotherapeuticintervention5,6.

Currently, the most effective method for measuringtelomerase activity is based on the telomeric repeatamplification protocol (TRAP) assay4,8. This methodconsists of a two-step in which telomerase first addstelomeric repeats onto the 3′-end of the substrate oligo-nucleotide (TS), and then in a second step, the extend-ed products are amplified by polymerase chain reac-tion (PCR) using TS and a reverse primer (RP), a pri-mer that is complementary to the telomeric repeats.The PCR products are then analyzed by polyacryl-amide gel electrophoresis (PAGE). Although TRAPassay is a very sensitive method by which telomeraseactivity in a small tissue sample or tumor biopsy canbe detected, the conventional TRAP procedure hassome disadvantages, particularly in the sample detec-tion and data analysis step: it is time-consuming toanalyze PCR amplicons by PAGE, and it is necessaryto measure the area or intensity of sixbase ladders bydensitometry with a computer program for quantitativeanalysis. To overcome these limitations, many adapta-

Original Research

Microchip-based capillary electrophoretic analysis oftelomerase activity for cancer diagnostics

Se Jin Kim1, Seok Jin Choi1, Rameshkumar Neelamegam1 & Tae Seok Seo1

Received: 10 October 2009 / Accepted: 14 November 2009 / Published online: 20 March 2010The Korean BioChip Society and Springer 2010

1Department of Chemical and Biomolecular Engineering(BK21 Program) and Institute for the BioCentury, KAIST, Daejeon 305-701, KoreaCorrespondence and requests for materials should be addressedto T.S. Seo ( seots@kaist.ac.kr)

tions of the conventional TRAP assay have been report-ed, such as TRAP scintillation proximity assay9, TRAP-ELISA10, TRAP hybridization protection assay11, fluo-rescent TRAP assay12,13, and stretch PCR assay14.ELISA and fluorescent assays have become the mostcommon methods for telomerase detection becausethey do not require radioactivity or gel electrophore-sis. However, these methods involve many multistepexperimental procedures, and precise quantificationand elimination of non-specific fluorescence back-ground are troublesome. Nevertheless, most of theTRAP systems use a slab gel-based electrophoresis tosize and quantify the tandem repeated PCR amplicons,which suffer from time- and labor-intensiveness andhard implementation into the clinical laboratory. Capil-lary electrophoresis (CE) has gained much attractionas a new separation technique by virtue of its highspeed, resolution, reproducibility and high degree ofautomation, which is particularly well suited for a widevariety of diagnostic applications. Atha and Hess etal. compared the efficiency of slab gel and capillaryelectrophoresis to screen the telomerase activity15,16.Their results showed that CE method would streamlinevalidation of the telomerase TRAP assay with theabove mentioned advantages over slab-gel electro-phoresis. In recent years, there has been considerableinterest in adapting electrophoresis technology tominiaturized microfluidic formats. The CE microchipbased analysis technique has several advantages overconventional large-scale counterparts, including highseparation efficiency, miniaturization, low sample/reagent consumption, and high-throughput capability.More importantly, these devices could be integratedwith other microfluidic units such that their function-ality and reliability could be greatly enhanced. Severalintegrated microfluidic systems, including CE/massspectrometry17, CE/optics18, and sample pretreatment/CE19 have been successfully reported.

In this report, our objective was to employ a capil-lary electrophoretic microdevice for the separation ofTRAP-based PCR amplicons and the screening of telo-merase activity in the cancer cells with high-speedand high sensitivity. To the best of our knowledge,this is the first report using the microchip-based capil-lary electrophoretic analysis for telomerase activityas cancer diagnostics.

Results and Discussion

Principle of the TRAP assay

Figure 1 illustrates the principle of the TRAP assaycomposed of telomeric repeat extension of a substrateoligonucleotide by telomerase, and PCR amplification

using Amplifluor® RP primers. The 41-nucleotide (nt)-long energy transfer (ET) reverse primer (Amplifluor®

RP primer) consists of a 3′-end sequence complemen-tary to the target sequence (telomeric repeats) and a5′-end hairpin structure. The hairpin of Amplifluor®

RP primer was labeled with a fluorescein as a donorand a DABCYL, a nonfluorescent chromophore, asan acceptor20. When the Amplifluor® RP primer isunincorporated and maintained as a hairpin confor-mation (the melting temperature [Tm] of the hairpinstructure in Amplifluor® RP was reported as 67 C-68 C21), the emission of the fluorescein linked to the5′-end of the primer is quenched by the energy trans-fer from the fluorescein to the DABCYL due to theclose proximity by the intramolecular hairpin forma-tion. Upon linearizing to form a part of the amplifica-tion product, energy transfer between the fluorophoreand the quencher is prohibited since they are separatedby over 15 nt, resulting in an enhanced fluorescenceemission signal20,22.

Microfabricated capillary electrophoresis vs. PAGE

Due to the benefits of reducing sample consumption,faster genetic analysis as a result of diminished jouleheating in electrophoresis, and increased sensitivitydue to their typically smaller interrogation volumes,capillary electrophoretic microtechnologies have beenwidely used in analyzing biological molecules such

BioChip J. (2010) 4(1): 42-48 43

Figure 1. Schematic illustration for the principle of the TRAPassay.

Addition of telomeric repeats by telomeraseTS

TSQuencher

Fluorophore

Amplifluor® RP primer

TS

TS

Synthesis of complementary strand/1st cycle of PCR

PCR amplification

Accumulation of florescent TRAP product

+

as DNA sequencing23, short tandem repeat (STR)analysis24,25, and pathogen detection26,27. Figure 2 illus-trates the microfabricated CE design and operationused for diagnosis of telomerase activity of cancercells. The CE microdevice consists of a cross-injectorand a 6 cm long separation channel which is coveredwith a silicon rubber heater for denaturing conditions(Figure 2A). PCR products in the sample reservoirwere injected electrokinetically into the injection chan-nel toward the waste reservoir by applying 1,000 V atthe waste for 60 s while floating the cathode and theanode reservoirs. The injected sample plug (~2 nL)located at the intersection was separated at 260 V/cmwith a backbias voltage of 500 V at the sample andthe waste reservoirs for 10 s to prevent sample bleed-ing. Subsequently, the separation proceeded along witha 6 cm long channel at 225 V/cm with the sample andthe waste reservoirs floated (Figure 2B). The entireCE procedure was complete within 5 min. On the otherhand, the conventional gel-based method such asPAGE, it takes 3 hours to prepare the gel system andseparate the PCR products. In addition, more than3000-fold volume of reagents such as acrylamide andbuffer was used for the gel preparation, and 100-foldexcess DNA products are necessary for sample load-ing. The weak trace of peaks was not distinguished asclear as a micro-CE analysis, demonstrating the supe-

rior speed, sensitivity and resolution of microchip-based CE analysis to the conventional PAGE.

Limit of detection for telomerase activity screening

Detection limit of telomerase activity is an importantissue in diagnosing cancer, because the amount of telo-merase activity might be correlated with the degreeof cellular immortality. In particular, the capability ofcancer screening with low cell numbers would beprecious in case of cell scarcity or fast analysis with-out culturing steps in a clinical laboratory. To investi-gate the detection limit of telomerase activity on a CEmicrochip, we performed the protein extraction fromthe 106 positive cancer cells, and serially diluted theextract for TRAP-PCR experiments. Considering thedilution factor, the added quantity of proteins for PCRcocktails is equivalent to the cell number ranging from5 to 5,000. For a negative control, TRAPeze® 1CHAPS lysis buffer was added to PCR reaction mix-ture instead of cell extracts. Figure 3 shows electro-pherograms of PCR amplicons separated on a micro-chip. Typical pattern displays that the first PCR ampli-con peak (61 bp) is highest, and then 6-bp repeatedpeak intensities are gradually decreased with a regularinterval due to the preferential amplification efficiencyof smaller sized DNA. Such peak patterns were obtain-

44 BioChip J. (2010) 4(1): 42-48

Figure 2. (A) Schematic of a microfabricated capillary electrophoretic device. (B) Capillary electrophoresis operation procedure(left panel) and the corresponding real-time fluorescence images in a microfluidic channel (right panel).

Cathode (C)

A. Chip design B. CE operation procedure

1. Injection

2. Backbiasing

3. Separation

S (0 V)

S (500 V)

C (0 V)

C (0 V)

W (1000 V)

A (1800 V)

A (2100 V)

W (500 V)

C (Floating) Injection

Backbiasing

Separation

S (Floating)

Flow

W (Floating)

A (Floating)

Flow

Waste (W)

Anode (A)

Sample (S)

Silicon rubber

Imaging region

PDMS

ed for all the samples and no telomerase activity couldbe detected for the negative control, demonstratingthe limit of detection (LOD) of telomerase activity ona micro CE is 5 cells.

Detection of telomerase activity in various humancells

Since the successful telomerase activity detection ona CE chip using positive cells was demonstrated, wefurther performed the telomerase activity screeningon a microfabricated CE detection system using vari-ous human cancer cell lines, namely, A549, SK-N-SH, and MCF-7. Same number (5000 cells) of eachcell was subjected to the TRAP PCR reaction mixtureand carried out for the telomerase extension and PCRamplification. Peak pattern was quite similar to thatof positive cells, and all the peaks of the PCR ampli-fied products were resolved on a chip-based CE methodwithin 5 min as shown in the electropherogram ofFigure 4. These results clearly confirmed the presenceof telomerase activity in all immortal cancer cells.

Therefore, this result gave us clear evidence that themicrofabricated CE system is very fast and sensitivetechniques as a cancer diagnostic tool to determinethe telomerase activity across different cell lines. How-ever, the peak intensity varies depending on the cancercell lines, implying that the telomerase quantity andactivity could be different among cell types. Due toits high telomerase activity, we chose MCF-7 cell fora dilution factor study. Similar to the LOD test of con-trol cells, we serially diluted the MCF-7 cells from5000 to 1, and the resultant CE data on a chip weredisplayed in Figure 5A. Although a single cell pro-duced a minor peak at 61 bp, the 6-bp telomeric repeatamplicons were not clearly detected. In the case offive cell input, the repeated pattern in the electrophero-gram was more obvious. As the cell number increased,the first peak at 61 bp became dominant, and the sub-sequent telomeric peaks with 6-bp interval were repre-sented with gradually decreased intensities. The sum-mation of the peak areas divided by an internal stan-dard peak proportionally correlated with the logarithmscale of input cell numbers as shown in Figure 5B.These results demonstrated that we could identify thetelomerase activity of MCF-7 cancer cells with a limitof detection of 5 cells, so that the potential of a rapidand high sensitive cancer diagnostics on a chip is pro-mising.

Conclusions

We have developed a TRAP-microfabricated CE

BioChip J. (2010) 4(1): 42-48 45

Figure 3. Electropherograms of a limit of detection test fortelomerase activity screening using positive cells on a microchip.

Rel

ativ

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cenc

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tens

ity

Primer

Negative control

5 cells

50 cells

500 cells

5000 cells

PCR product

150 200 250

Time (s)

Figure 4. Electropherograms of PCR amplicons generatedby the TRAP/CE assay using cancer cell lines.

Rel

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ity150 200 250

Time (s)

Primer MCF-7

A549

SK-N-SH

method for analysis of telomerase activity with highspeed, improved sensitivity, and reproducibility. Telo-merase activity was a useful genetic marker for cancer

diagnostics, and was identified with a detection limitof 5 cells. With an appropriate robotic implementationand expansion into an ultra-dense CE array, the cancergenetic CE analysis could be performed in an auto-matic and high-throughput manner. Furthermore, theCE microchip can be integrated with a PCR chamberas well as a cancer cell preparation step on a singlewafer which is under way in our laboratory. The fullyintegrated TRAP microdevice will realize rapid, pre-cise, and on-site telomerase activity analysis, enablingcancer diagnostics in the clinical laboratories.

Materials and Methods

Cell culture and protein extraction

The human cancer cell lines were derived from theepithelial cells of lung adenocarcinoma (A549, ATCCCCL-185), neuroblastoma cells (SK-N-SH, ATCCHTB-11) and breast cancer cells (MCF-7, ATCC HTB-22). Growth properties of all cell lines used in thisstudy are as adherent cells and used between subcul-ture passages 20 and 30. The cell lines were maintain-ed in Dulbecco’s-Modified Eagle’s Medium (DMEMfor A549 and MCF-7) and Minimum Essential Medium(MEM for SK-N-SH) containing 10% fetal bovineserum (FBS) and 1% penicillin (10,000 IU/mL)-strep-tomycin (10,000 μg/mL). Subsequently, cells weregrown and maintained in a T-75 cell culture flask at37 C in a CO2 humidified incubator with 80-90% con-fluence before cell detaching and subculture. Afterthe process of phosphate buffered saline (PBS) wash-ing, cells were incubated in 3 mL of 0.2% trypsin-EDTA for 3-5 min at 37 C in CO2 incubator. Cell sus-pensions were centrifuged for 3 min at 1,300 rpm. Thepellet was re-suspended with fresh medium and theseed density was adjusted using a hemocytometerbased cell counting with the aid of an inverted micro-scope (Nikon SMZ 1500 microscope, Tokyo, Japan).A certain number of cells were collected for proteinextraction to perform TRAP-PCR assay. Telomerasepositive cells were used from the TRAPeze® XL Telo-merase Detection Kit (S7707, Millipore, Billerica,MA, USA).

For extraction of telomerase enzymes from the cells,the cells were washed once with PBS buffer and cen-trifuged at 1,300 rpm for 3 min to remove the buffer.Then, the cells were resuspended in 200 μL of 1CHAPS lysis buffer provided in the kit and incubatedon ice flakes for 30 min according to TRAPeze® XLTelomerase Detection Kit protocol. After spinning thesuspension at 12,000 g for 20 min at 4 C, only 160 μLof the supernatant was collected and its protein con-centration was determined by NanoDrop spectrometer

46 BioChip J. (2010) 4(1): 42-48

Figure 5. (A) LOD study of TRAP/CE assay using MCF-7cancer cell line, and (B) the quantitative analysis data.

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1 10 100 1000

Cell equivalents (Log scale)

(A)

(B)

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(ND-1000, NanoDrop products, Wilmington, DE,USA).

TRAP-PCR assay

For PCR amplification reaction, cell extracts obtainedfrom A549, SK-N-SH, MCF-7, and positive cells weremixed with H2O, 2 U of FastStart Taq DNA polymerase(12032902001, Roche Applied Science, Indianapolis,IN, USA) and 5 TRAPeze® XL Reaction Mix con-taining telomerase substrate oligonucleotide (TS pri-mer), RP Amplifluor® primer, and dNTPs in a buffer(100 mM Tris-HCl, pH 8.3, 7.5 mM MgCl2, 315 mMKCl, 0.25 mM Tween 20, 5 mM EDTA, 0.5 mg/mLBSA) in a final volume of 50 μL. Using a thermocycler(DNA Engine®, Bio-Rad), telomere extension stepwas carried out at 30 C for 30 min and PCR amplifi-cation was followed by thermal cycling steps: 95 C for6 min (one time at first), 94 C for 30 s, 59 C for 60 s,72 C for 60 s for 36 cycles. After the PCR reaction,all the samples were electrophoresed in the capillarymicrochannel to detect telomerase activity for eachcell line. For a limit of detection study, the cell extractwas isolated from 106 positive cells and then seriallydiluted which corresponds to the cell numbers from 5to 5,000 used for TRAP-PCR reactions.

Fabrication of CE microdevice

To form micro-sized channels on the borofloat glasswafer (100 mm diameter, 1.1 mm thickness, PG & O,Santa Ana, CA, USA), 200 nm thick amorphous sili-con was coated on the glass wafer using low-pressurechemical vapor deposition (LPCVD). After hexame-thyldisilazane (HMDS, Sigma-Aldrich, USA) vaporpriming for 15 min, photoresist was spincoated on theamorphous silicon wafer with 2 μm thickness. Themicrochannel patterns were transferred to the photo-resist layer by UV exposure through the chrome mask.After developing the photoresist layer, the exposedsilicon layer was removed by reactive ion etching(RIE) in SF6 plasma. Concentrated hydrofluoric acid(49% HF) is used to etch the glass at an average rateof 7 μm/min to form the microfluidic channels with180 μm width and 50 μm depth. The remains of photo-resist were washed with acetone for 10 min, and thesilicon layer was then removed by RIE in SF6 plasma.The 1-mm diameter holes were drilled using SherlineVertical milling machine (Model 2010, Sherline Pro-ducts, Inc., Vista, CA, USA). The patterned wafer wasthermally bonded to a blank borofloat glass at the668 C for 4 hr. To set up electrode connection to thereservoir, 3 mm-diameter punctuated PDMS membrane(3 mm thickness) was treated in a UV-ozone cleanerfor 6 min, and then assembled on the glass holes.

Operation of CE microdevice

Prior to CE operation, the microchannels were firstcleaned using piranha (3 : 1 H2SO4/H2O2) solution for15 min. After extensive H2O washing, the channelswere pretreated with a 50% dynamic coating (DEH-100, The Gel Co., San Francisco, CA, USA) in metha-nol for 15 min to suppress electroosmotic flow. A 5%(w/v) linear polyacrylamide (LPA) with 6 M urea in 1

Tris TAPS EDTA (TTE) was injected as a separa-tion sieving matrix, from the anode hole using a 1 mLdisposal syringe to fill the channels. The separationchannels (effective length: 6 cm) were heated at 70 Cwith a silicon rubber heater (SR020312, Hanil ElectricHeat Engineering Co., Korea) for denaturing condi-tions. After loading 10 μL of TTE buffer into the cath-ode, anode, and waste reservoirs, 7 μL of PCR productin the sample well were electrophoresed through thethree steps (injection, backbiasing, separation) as shownin Figure 2. The fluorescence signal of separatedPCR amplicons was detected at the end of the separa-tion channels with a laser-induced confocal fluores-cence microscope (C1si, Nikon, Japan). A 0.016 mm2

detection area was scanned with the speed of 5 frame/sfor data acquisition. For excitation of the fluoresceindye, a wavelength of 488 nm from an argon laser wasused with a power intensity of 3.6 mW from the objec-tive.

Acknowledgements This research was supported byBasic Science Research Program through the NationalResearch Foundation of Korea (NRF) funded by the Mini-stry of Education, Science and Technology (2008-0060721& No. R11-2001-089-10002-0), and also partially by Tech-nology Development Program for Agriculture and Fore-stry, Ministry for Agriculture, Forestry and Fisheries,Republic of Korea.

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