Targeted isolation and analysis of single tumor cells with aptamer-encodedmicrowell array on microfluidic device{

Qiushui Chen, Jing Wu, Yandong Zhang, Zhen Lin and Jin-Ming Lin*

Received 27th July 2012, Accepted 24th September 2012

DOI: 10.1039/c2lc40858a

Microfluidic-based single cells analysis has been of great interest in recent years, promising disease

diagnosis and personalized medicine. Current technologies are challenging in bioselectively isolating

specific single cells from complex matrices. Herein, a novel microfluidic platform integrated with cell-

recognizable aptamer-encoded microwells was specifically developed to isolate single tumor cells with

satisfied single-cell occupancy and unique bioselectivity. In this work, the designed microwell-

structures enable us to encourage strong 3D local topographic interactions of the target cell surface

with biomolecules and regulate the single-cell resolution. Under the optimized size of microwells, the

single-cell occupancy was significantly enhanced from 0.5% to 88.2% through the introduction of the

aptamer. Analysis of the target cells was directly performed in short time periods (,5.0 min) with

small volumes (4.5 mL). Importantly, such an aptamer-enabled microfluidic device shows an excellent

selectivity for target single cells isolation compared with three control cells. Subsequently, targeted

isolation and analysis of single tumor cells were demonstrated by using artificial complex cell samples

at simulated conditions, and various cellular carboxylesterases were studied by time-course

measurements of cellular fluorescence kinetics at individual-cell level. Thus, our technique will open

up a new opportunity in single-cell level-based disease diagnosis and personalize medicine screening.

Introduction

New diagnostic technologies at single-cell level have now

changed the understanding and clinical practice of oncology.1,2

The studies of cellular heterogeneity in proteomics and gene

expression will enable early diagnosis and personalized medi-

cine.3 Currently, single-cell analysis is emerging as a powerful

tool to reveal the cellular heterogeneity in cell populations at the

individual-cell level for understanding various single cells

signaling.4–6 Nowadays, flow cytometry is a traditional approach

to examine the cellular components of single cells with high-

throughput using fluorescence labeling.7,8 Alternatively, capil-

lary electrophoresis (CE) combined with laser-induced fluores-

cence (LIF),9 electrochemistry10 and chemiluminescence11 are

also widely utilized for single cells separation and analysis.

Despite such achievements, however, those analytical technolo-

gies make it difficult to provide information on both temporal

and spatial cellular responses.

Over the last decade, microfluidic technologies increasingly

play an important role in biological research, promising high-

throughput assays, fast analysis and low reagent require-

ments.12–16 Of particular interest is microfluidic single-cell

analysis that enables single-cell manipulations,17 long-time

culture,18 drug-related treatments19 and high-efficiency screen-

ing20 on temporal and spatial micro-scales. Microfluidic-based

single cells analysis, such as polymerase chain reaction,21,22 drug-

induced apoptosis19 and enzyme kinetics,23,24 shows great

potential for application in medical diagnosis and clinical

treatment. A number of approaches have been explored in the

isolation and operation for single cells, including physical

barriers,18,23,25 fluid mechanics,17,26–28 or gravity mechan-

isms.24,29,30 Although these strategies represent important

advances, much improvement is still needed. For example, the

approaches of micro-fabricated structures, such as microwell-

based docking,26,29,30 microdam-based locating23,31 and micro-

cavity-based trapping,32,33 which rely on the degree of physical

structure capture, are technically challenging in bioselectively

isolating single cells. Besides, the encapsulation of single cells and

reagents in independent aqueous microdroplets provided excel-

lent cellular microenvironments and increased throughputs,20,27

but showed limited capability to deal with complex cell samples

(e.g. cell suspensions and whole blood samples). Thus, targeted

isolation of single cells presents a tremendous interest. Given

these technical challenges, bio-recognizable microfabricated

physical structures will be a powerful strategy and open up

unique opportunities for isolating single specific cells.

It has been well-demonstrated that aptamers, a kind of nucleic

molecule (RNA or DNA), can bind to target tumor cells with

high specificity and affinity.34,35 Herein, we report a novel

Beijing Key Laboratory of Microanalytical Method and Instrumentation,Department of Chemistry, Tsinghua University, Beijing, 100084, China.E-mail: jmlin@mail.tsinghua.edu.cn; Fax: (+86) 10 62792343{ Electronic Supplementary Information (ESI) available: See DOI:10.1039/c2lc40858a

Lab on a Chip Dynamic Article Links

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microfluidic platform for the targeted isolation of single tumor

cells with aptamer-coated microwells based on the single-cell-

containable micro-chambers and their bio-specific interactions.

The specifically designed microwell-structures enable us to

encourage strong 3D local topographic interactions of the target

cell surface with biomolecules and regulate the single-cell

resolution (Fig. 1A and C). Such an aptamer-enabled micro-

fluidic device obtained unique bioselectivity and significantly

enhanced single-cell occupancy ranging from 0.5% to 88.2%

(Fig. 1A and B). Subsequently, targeted isolation and analysis of

single tumor cells were demonstrated by using artificial complex

cell samples at simulated conditions, and various cellular

carboxylesterases were temporally and spatially studied by

measuring cellular fluorescence kinetics at an individual-cell

level. Consequently, the development of the bio-selective single

cells isolation microchip platform serves two purposes. First, it

will permit us to separate specific tumor cells from complex

clinical samples, such as cell suspensions and whole blood

samples, allowing direct single-cell level-based disease diagnosis.

Second, it will enable a high-throughput assay of cellular enzyme

kinetics in individual tumor cells, promising personalized

medicine. We believe this aptamer-encoded microwell-based

microchip will represent a novel platform for single-cell analysis

and offer new opportunities in clinical application.

Experimental

Materials and chemicals

Dulbecco’s modified Eagle’s minimal essential medium (DMEM,

Gibco, Grand Island, NY), RPMI medium 1640 (Gibco, Grand

Island, NY), trypsin EDTA (Gibco, Grand Island, NY), sodium

pyruvate (100 mM) and calcein AM were purchased from

Invitrogen (CA, USA). Chromium layer patterned glass sub-

strate was fabricated by Qingyi Precision Maskmaking Co., Ltd.

(Shenzhen, China). Silicon wafers were purchased from Xilika

Crystal Polishing Material Co., Ltd. (Tianjin, China). Negative

photoresist (SU-8 2050) and developer were purchased from

Microchem Corp. (Newton, MA). Poly-dimethylsiloxane

(PDMS) and the curing agent were obtained from Dow

Corning (Sylgard 184, Midland, MI, USA). Avidin and

Phosphate Buffered Saline (PBS, pH = 7.4) were purchased

from Sigma-Aldrich Company. The aptamer was synthesized by

Sangon Biotech Co., Ltd. TD05: 59-AAC ACC GGG AGG

ATA GTT CGG TGG ATG TTC AGG GTC TCC TCC CGG

TGT TTT TTT TTT-39-Biotin. All other reagents were of

analytical reagent grade and used without further purification.

Cell culture and preparation

All cancer cell lines (Ramos cells, CCRF-CEM cells, MCF-7

cells and HepG2 cells) were obtained from the Cancer Institute &

Hospital, Chinese Academy of Medical Science. Ramos and

CCRF-CEM cells were cultured in RPMI medium 1640

supplemented with 10% FBS and 100 IU mL21 penicillin–

streptomycin. HepG2 and MCF-7 cells were cultured in DMEM

media with 10% FBS and 100 IU mL21 penicillin–streptomycin.

Specially, 0.11 g L21 sodium pyruvate was added to RPMI

medium 1640 for CCRF-CEM cells culture. Both cell lines were

cultured in a 5% CO2-humidified air atmosphere at 37 uC and

maintained by passaging twice weekly. The MCF-7 and HepG2

cells were dissociated from the culture dish with 0.25% Trypsin

EDTA in exponential growth phase. In the experiments, the cells

were centrifugated 5.0 min at 1000 rpm after being rinsed with

PBS buffer, and a cell concentration of 5.0 6 107 cells mL21 was

used for single-cell capture.

Fabrication of the microwell-structured array on a microfluidic

device

The microfluidic chip was fabricated using standard soft

lithography and replica molding techniques (see the details in

the Supporting Information{).36 The fabrication of the micro-

well array on the glass substrate was carried out by using the

chromium layer as its photomask. The glass substrate was then

cut to pieces of 2.5 cm 6 2.5 cm. The chromium image was

designed using AutoCAD software with 18 000 (120 6 150)

Fig. 1 A schematic illustration of the microfluidic platform used for single target cells isolation. (A) The microfluidic device consists of a PDMS layer

with microchannels and a glass substrate with microwells. (B) The three-dimensional topography of the microwell array was characterized by a

scanning electron microscope (SEM). (C) Two steps for glass surface functionalization were conducted by the deposition of avidin and immobilization

of biotin–aptamer.

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adjacent spot (total space: 2.1 mm 6 2.4 mm). The microwell

array on the glass substrate was etched by hydrofluoric acid/

ammonium fluoride (HF/NH4F, 5.0 M/5.0 M) solution for about

2.0 min. Four different sizes of microwell structure with

diameter/depth were fabricated at 18 mm/5.0 mm, 20 mm/5.0

mm, 22 mm/5.0 mm and 20 mm/8.0 mm, respectively. The microwell

sizes can be easily controlled by the etching time with the help of

an optical microscope. After washing with deionized water, the

glass substrate was soaked in chromium etchant for 5.0 min to

remove the patterned chromium layer. The prepared microwell

array on glass substrate was then cleaned by piranha solution

(H2SO4:30% H2O2 = 3 : 1, v/v). The microwell structure was

characterized by a scanning electron microscope (SEM). Non-

covalent bonding between the PDMS layer and the glass slide

was conducted using two clamps for recycling the glass slide with

the microwell array (see Fig. S1){.

Aptamer-encoded microwell array

A key experimental detail was to keep the microwell array on

the glass substrate hydrophilic to reduce nonspecific adhesion

of cells. This was achieved by cleaning the microwell array using

piranha solution (H2SO4:30% H2O2 = 3 : 1, v/v) in 100 uC to

form a hydrophilic interface each time. After rinsing with

deionized water three times, the glass slide was dried under

nitrogen gas at room temperature, but not at a high

temperature. Then, the microwell array was covered with a

PDMS layer with two microchannels, and bubbles in the

microchannel were removed by sampling 50 mL PBS buffer

(10 mM, pH = 7.4). Avidin (1.0 mg ml21, 5.0 mL) in PBS buffer

was first incubated in the microwells for 5.0 min and rinsed with

5.0 mL PBS buffer three times. Finally, biotin–aptamer (5.0 mL,

100 mM) in tris-EDTA (TE) buffer was incubated for 5.0 min

and rinsed with 5.0 mL PBS buffer three times. The prepared

microdevice was kept at 4 uC. Tapping mode atomic force

microscopy (AFM) was conducted to characterize the immobi-

lization of avidin and aptamer on the glass substrate (see

Fig. S2){.

Isolation of single cells and optimizing conditions

In the experiments, a cell concentration of 5.0 6 107 cells mL21

was rinsed with PBS buffer. To study the effect of aptamer

concentrations on single cells occupancy, a series of concentra-

tions (0, 25, 50, 75, 100 mM) was prepared to be immobilized on

the microwells. To study the effect of microwell size on

single cells occupancy, single cells isolation was conducted on

four different sizes of microwells. In all of these experiments, 4.5

mL Ramos cells were used as the target cells by a transfer

pipette. To study the selectivity of single cells occupancy,

Ramos cells (lymphoma cancer, target cells), CCRF-CEM cells

(leukemic lymphoblasts, control), MCF-7 cells (breast cancer,

control) and HepG2 cells (liver cancer, control) in PBS buffer

were introduced to microchannels under static conditions. After

1.0 min of cells capture, the microchannel was washed three

times with PBS buffer and observed under a fluorescence

microscope. The captured single cells can be easily counted

by image analysis software (Image-Pro plus 6.0, Media

Cybernetics, USA).

Analysis of single-cell enzyme kinetics

An artificial mixture of cells (Ramos cells and CEM cells, 1 : 1)

was used as complex biological samples. A cell concentration of

5.0 6 107 cells mL21 was rinsed with PBS buffer and then

introduced to the microchannel for selective single cells isolation.

10 mL PBS buffer was used to rinse the microchannel after 1.0

min. The single cells in the microfluidic device were stored in a

humidified 5% CO2 environment at 37 uC. To perform time-

course enzyme kinetics analysis, this microdevice was placed on

the platform of a fluorescence microscope. 4.5 mL of 4.0 mM

calcein AM in PBS solution was introduced into the micro-

channel. The intracellular fluorescence was monitored by a

fluorescence microscope at the wavelength of 488 nm for 10 min.

This fluorescence microscope was equipped with a cooled CCD

camera (DFX 3000) and software of Leica Application Suite,

LAS V2.7. A series of images (1 s exposure) were acquired at 10 s

intervals.

Results and discussion

Design and operation of the aptamer-immobilized microwell-

structured microdevice

The designed microdevice consists of two components: an

overlaid polydimethysiloxane (PDMS) layer with two micro-

fluidic channels and a glass substrate with aptamer patterned

microwell array (Fig. 2A and B and Fig. S1a{). Previous reports

have confirmed that the low height of the microchannels (60 mm

in this case) will encourage cell-substrate contact frequency,

which increases cell capture efficiency.35,37,38 The designed

microchannels were crucial to controlling the cell microenviron-

ment, low reagent consumption and repeatable high-throughput

assay. The microwell-structured substrate was fabricated by

using a lithographic method and chemical etching process. A

diameter of 18–22 mm and depth of approximately 5.0 mm and

8.0 mm were chemically etched onto the chromium-patterned

glass substrate, yielding a feature of density of 3500 wells per

mm2 (Fig. 2C). According to previous methods,34 an avidin

coating (4.5 mL, 1.0 mg ml21) was introduced onto microwell-

structured glass substrates for 5.0 min (Fig. 2D). Prior to the

Fig. 2 A schematic illustration of the microfluidic platform used for

single target cells isolation. (A), (B) The microfluidic device consists of a

PDMS layer with microchannel and a glass substrate with microwells.

(C) The three-dimensional topography of the microwell array was

characterized by a scanning electron microscope (SEM). (D) Two steps

for glass surface functionalization were conducted by the deposition of

avidin and the immobilization of biotin–aptamer.

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single cells capture experiments, 4.5 mL of biotinylated aptamer

was conducted onto the avidin-coated substrates in each

microchannel. Accordingly, single target cells will be selectively

captured by the aptamer coated microwells from the complex cell

samples, which is attributed to the fitted 3D local topographic

interactions between the cell surface and aptamer on microwells

(Fig. 1A). On the contrary, the microwell array without an

aptamer coating will lose the ability of isolating single cells owing

to a lack of bio-specific interactions with target cells (Fig. 1B).

Typically, the microwell structure is crucial to regulate the single-

cell resolution (Fig. 1C). The whole microfluidic system

integrated with an aptamer-coated microwell array, therefore,

can be designed for selectively isolating single target cells and

high-throughput single cell assays.

Single cells capture with aptamer-encoded microwell-structured

micro-platform

The performance of single-cell analysis in this integrated

microdevice was first demonstrated by isolating leukemia cells

(Ramos cells) that functioned as target cancer cells. An optimal

cell concentration (5 6 107 cells per mL) was determined

according to the resulting single-cell occupancy at static cell-

capture conditions. As shown in Fig. 3A, the aptamer-

immobilized microwell enables high single-cell occupancy

(percentage of microwells containing single cells). As a control,

microwell-structured substrates without any surface modifica-

tion and only modified with avidin were also examined in

parallel. The single cells capture yield was only 1.4% and 0.5% on

microwell-structured substrates and avidin-modified substrates,

as opposed to 88.2% on aptamer-immobilized microwell-

structured substrates. This result suggests that aptamer-enabled

microwells are possibly responsible for the high single cells

occupancy (Fig. 3B). This is reasonable because the 3D-

structured microwells exhibited obvious morphology differences

in the target cells capture: i) the microwells substrates with 3D-

structure is more matched with the spherical cells and thereby

creates more recognition sites between the surface antigen of the

cell membrane and the aptamer immobilized on the microwells,

which yields strong 3D local topographic interactions for single

cells capture;37 ii) the structure of microwells is playing an

important role in regulating the single-cell resolution.

Consequently, these evidences indicated that the integrated

aptamer-encoded microwell-structured substrate in the micro-

device was a novel single-cell-capture platform for biological

application.

Effect of aptamer concentration and microwell-size on single cells

occupancy

Since the aptamer shows excellent capability in target cells

capture, the concentration of aptamer was reported to directly

relate to its capture efficiency.35 To investigate this effect,

different concentrations of aptamer were immobilized onto the

microwell-structured glass substrate. As shown in Fig. 4A, the

single cells occupancy was obviously increasing with the

increased concentrations of aptamer, ranging from 0 to 100

mM. In our experiments, the single cells occupancy was as high as

88.2%, compared with the control (no aptamer immobilization)

of 0.5%. Therefore, the results also demonstrated that the

immobilization of aptamer plays an important role in the

performance of single cells capture.

To capture and analyze single cells with high-efficiency, the

optimal geometry of microwells was conducted with the

diameters of 18, 20 and 22 mm at the depth of 5.0 mm and 8.0

mm (Fig. 4B). When the microwells were 18 mm in diameter, most

of the microwells were filled with target cells at single-cell

resolution (occupancy 88.2%). However, the single cells occu-

pancy was decreasing with increased diameter of the microwells

at 20 and 22 mm with the deposition of two cells. When the

microwells were at the diameter of 20 mm, the single cells

occupancy was achieved in different depths at 5.0 mm and 8.0

mm. It shows that the single cell occupancy of 5.0 mm depth will

be a little higher than that of 8.0 mm depth; these results are

reasonable, because the larger volume will lead to a high

probability of double cells per microwell. Thus, the microwells

with 5.0 mm depth and 18 mm diameter were finally selected for

further experiments. All of the single-cell occupancies under

Fig. 3 The performance of single Ramos cells onto the microwell array.

(A) Bright field images of single cells captured onto the aptamer-coated

microwells and the non-modified microwell array as a control. (B)

Quantitative evaluations of single-cell occupancy were performed under

control, avidin-coated microwell array and avidin/biotin–aptamer-coated

microwell array, respectively. Avidin: 1.0 mg ml21, Biotin–aptamer: 100

mM.

Fig. 4 The effect of aptamer concentrations and microwell sizes on

single-cell occupancy. (A) The single Ramos cells occupancy was raised

with increasing aptamer concentrations immobilized in the microwell

array. The concentrations were studied in 0, 25, 50, 75, 100 mM. (B)

Ramos cells occupancy was conducted under different sizes (diameter

and depth) of aptamer-encoded microwells. Concentration of aptamer:

100 mM. Error bars show standard deviations (n = 6).

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different sizes of microwells are listed in Table 1. In brief, the

diameter of the microwell was an important factor for high

single-cell occupancy, and the designed microwell array was

responsible for regulating the single-cell resolution.

Selectivity of single cells isolation from multiple-types of cancer

cells

Although a number of approaches have been developed to

isolate single cells for biological studies, they are always lacking

the ability to capture specific target cells for single-cell analysis.

Here, the selectivity of single-cell capture on the aptamer-

immobilized microwell-structured substrates was clearly demon-

strated by the target cells and three kinds of control cells. An

average of 88.2% single-cell occupancy was achieved by using the

Ramos cells as target cells. For comparison, other cancer-cell

lines (i.e., CEM, MCF-7 & HepG2 cancer cell lines, representing

both suspension and adherent cell types, respectively) were

employed as controls. Fig. 5 shows the low single-cell occupancy

of these three cancer-cell lines, that is, the aptamer-encoded

microwell-structured substrates are only capturing specific single

Ramos cells (target cancer-cell line). Our approach significantly

improved selectivity in target single cells capture that is distinct

from traditional physical methods.17,18,20,23–33 Thereby, this

fabricated microfluidic platform shows excellent performance

in selectively isolating single cells that will be potentially applied

in complex biological samples, which is promising for disease

diagnosis and drug assay at the single-cell level.

Enzyme kinetic analysis at single-cell level

Targeted isolation of single cells will enable its application in

complex biological samples and is thus promising for monitoring

drug-related intracellular responses at individual-cell level before

pharmacological treatment. For example, various carboxyles-

terases between individual cells hold essential biochemical

functions of aster and amide hydrolysis that are crucial to

activate antidrugs and inactivate other pharmaceuticals.39 To

establish this concept, artificial complex biological samples were

prepared by spiking lymphoma cells (target tumor cells: Ramos

cells) with an aliquot of CEM cells in media solutions. Our

approach resulted in isolating single Ramos cells with excellent

selectivity, providing the possibility of high-throughput single

cells analysis. Further, calcein AM was used as a substrate for

carboxylesterase that generates intracellular green fluorescence,

and the study of enzyme kinetic activity at individual-cell level

was achieved by time-course fluorescence analysis in further

experiments. As shown in Fig. 6A, the fluorescence intensities of

each single-cell increased as a function of time. The kinetic

curves for each single-cell present the different amounts of

intracellular carboxylesterase, revealing the diversity of condi-

tions or viability in target single cells that are associated with

cancers (Fig. 6B). This phenomenon is reasonable because of the

variations in cellular carboxylesterase expression levels between

individual cells.23,39 Based on this strategy, drug dosages and

combinations of therapies could be patient-defined to treat

individual disease in a clinical setting, thus providing the

possibility of developing personalized medicine.

Table 1 Single-cell occupancies under different sizes of microwells

Diameter (D) /depth (H) D = 18 mm/H = 5 mm D = 20 mm/H = 5 mm D = 22 mm/H = 5 mm D = 20 mm/H = 8 mm

Single-cell per microwell (%) 88.2 62.8 46.4 52.6SD (%) ¡3.4 ¡4.4 ¡7.3 ¡8.7Two cells per microwell (%) 7.5 10.7 21.7 14.1SD (%) ¡1.7 ¡2.5 ¡2.1 ¡2.0

Fig. 5 Investigation of single-cell capture selectivity on aptamer-

encoded microwells using Ramos cells (target cells), HepG2 cells

(control), CEM cells (control) and MCF-7 cells (control). Aptamer

concentration: 100 mM. Cell concentration: 5 6 107 cells mL21.

Fig. 6 Time-course measurements of carboxylesterase dynamic

responses to 10 mM calcein-AM at individual cells by a fluorescence

microscope. (A) The fluorescence images were recorded using a cooled

CCD camera at a 406 objective. (B) Relative fluorescence intensities of

single cells are increased as a function of time.

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Conclusion

In summary, a novel single-cell capture platform based on an

aptamer-encoded microwell-structured microdevice, described

here, was demonstrated to exhibit excellent selectivity. Our results

indicated that the highly-efficient single-cell occupancy was

obviously dependent on the concentrations of aptamers and the

size of the microwells. The 3D-structured microwell can also

encourage the interactions of the target cell surface with the DNA-

aptamer, resulting in a satisfactory single-cell occupancy

(y88.2%). The performance of single cells isolation with bio-

selectivity is distinct from existing technologies. Finally, the

isolation of single cells from the artificial complex of biological

samples was further applied to enzyme kinetic analysis on an

individual-cell level. We believe that the aptamer-enabled

selectivity of single cells isolation technology will open up

opportunities for the isolation of single tumor cells and for

personalized medicine based on a single-cell level, which is

promising for biological and medical application.

Acknowledgements

This work was supported by the National Natural Science

Foundation of China (Nos. 20935002).

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