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3 Immobilization of Human Cells on Slides and Subsequent Immuno-RCA

3 Immobilization of Human Cells on Slides and Subsequent Immuno-RCA

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S. Sarkar et al.

Fig. 8.1 (a) Schematic of RCA strategy on slides. (b, c) Detection of individual RCA amplicons

on glass slide coated with (b) 102/sq. mm molecules and (c) 101/sq. mm molecules of DNA

minicircle bound to DNA tag primer. Reproduced with permission (Konry et al. 2011). Copyright

Wiley-VCH Verlag GmbH & Co. KGaA

were washed three times for 2 min in 50 mL of Phosphate Buffered Saline (PBS).

The cells were then incubated with biotinylated anti-EpCAM Abs (1 μg/mL, R&D

systems (BAF960)) diluted in binding buffer (PBS, 1 % BSA 0.2 % Tween-20) for

45 min at RT. After washing, the slides were incubated with streptavidin (1 μg/mL)

for 45 min at RT. 1 µg/mL of the biotinylated primer was applied to the slide. Lastly,

the padlock probe (108 circles) was added and circularized by T4 DNA ligase in the

hybrid duplex with the primer (see Fig. 8.2a).


Ultrasensitive Isothermal Detection of Protein Analytes Using Rolling Circle…


Fig. 8.2 Illustration of EpCAM detection on PC3 cell surface. (a) (1) Binding of biotinylated Ab

to EpCAM molecule; (2) conjugation of biotin-DNA primer to the biotinylated Ab via avidin

bond; (3) hybridization of DNA circular probe; (4) RCA and fluorescent detection with Cy3 dye

(orange-red color). (b) EpCAM detection on PC3 cells immobilized on glass slide and counterstained with DAPI (blue color): (1) direct immunoassay test with anti-EpCAM Ab (no RCA); (2,

3) Signal amplification by RCA. (c) Microfluidic device design. (d) (I) Droplet generation junction; (II) RCA-generated bright spots observed in droplets on surfaces of single cells. Reproduced

with permission (Konry et al. 2011). Copyright Wiley-VCH Verlag GmbH & Co. KGaA

The RCA reaction mix consisted of 1× phi29 DNA polymerase buffer, 10 units

phi29 DNA polymerase (New England Biolabs), 200 μM dNTPs, and a 40 μM of

Cy3-dCTP (GE Healthcare). This mix (50 μL) was dropped on the slide, which was

then covered with a cover slip and sealed with rubber cement. The RCA reaction

was performed at 37 °C, for 1.5 h in a humidifying chamber. Then the slides were


S. Sarkar et al.

washed twice in buffer A (100 mM TrisHCl, 150 mM NaCl, and 0.05 % Tween20)

and once in 4× T buffer (4× SSC, 0.05 % Tween 20). After washing, a drop of

mounting medium with counterstaining DAPI (4,6-diamidino-2-phenylindole) at

50 mg/mL in PBS was added. Slides were then re-covered with a cover slip, and

after standing at RT for 5 min, the cover slip was secured by using nail polish.

In the direct immunoassay test (control experiment without RCA), the PC3 cells

were labeled with biotinylated anti-EpCAM antibodies and then detected with Cy3fluorescence-labeled streptavidin. The cells were also counterstained by DAPI contained in the mounting medium. Note that although DAPI is most used for selective

staining of cell nuclei, the whole PC3 cells fixed on slides are well stained with this

dye, too (Ge et al. 2015).


Droplet-Based Immuno-RCA and Imaging

For droplet-based RCA, 105 PC3 cells/mL were labeled with biotinylated primer

and DNA circular padlock (in a similar way as described above for immobilized

cells) in RCA reaction mixture (1× phi29DNA polymerase buffer, 10 units phi29

DNA polymerase, 200 μM dNTPs, and 40 μM of Cy3-dCTP fluorescently labeled

dCTP) and added to the microfluidic device. Following single-cell encapsulation,

the droplets were incubated in the channel for 1.5 h at 37 °C to allow RCA reaction.

Fluorescence images of droplets were captured on a Zeiss 200 Axiovert microscope

using an AxioCAM MRm digital camera and appropriate filter sets for Cy3.

Individual channels were imaged and overlaid for comparison. ImageJ software was

utilized for all image processing and analysis.


Optical Encoding of Microspheres

Amine-functionalized microspheres of 3.1 μM diameter were washed thrice with

PBS and then washed thrice again with 200 μL tetrahydrofuran (THF). Then,

200 μL aliquots of 0.1 or 0.5 M Europium (III) thenoyltrifluoroacetonate trihydrate

(Eu-dye) in THF were added for low- and high-intensity encoding of microspheres,

respectively. The two sets of microsphere suspensions were agitated in the dark for

2 h at RT. The microspheres were subsequently washed six times with 200 μL

MeOH followed by PBS washes. These low- and high-intensity Eu-encoded microspheres were stored in PBS supplemented with 0.01 % Tween-20 at 4 °C.


Functionalization of Microspheres

The suspensions of encoded microspheres (100 μL aliquots) were washed, added to

8 % glutaraldehyde solution, and shaken at RT for 2 h in the dark. After removing

excess glutaraldehyde, 45.3 μg of anti-IL-6 (clone 6708) or anti-IL-8 (clone 6217)


Ultrasensitive Isothermal Detection of Protein Analytes Using Rolling Circle…


monoclonal Abs (R&D Systems) were incubated with the glutaraldehyde-treated

microspheres in 500 μL PBS on a shaker at RT for 4 h. The Ab-coated microspheres

were treated with 300 μL Tris-Starting Block (TBS) blocking buffer (Thermo

Scientific) for 30 min in the dark and were stored in the TBS blocking buffer at 4 °C.


Fabrication of Arrayed Optical Sensor and RCA

on Functionalized Microspheres

A fiber-optic microarray was prepared by successively polishing the ends of the

optical fiber bundles with 30, 15, 6, 3, 1, 0.5, and 0.05 μM lapping films. Any

remaining residues were removed by sonicating the fiber bundles in water for 10 s.

The edge of the polished fiber was chemically etched to fabricate the rectangulararranged microwells. The etched fibers were carefully washed with Nanopure water

and treated with 200 μL Protein-free (PBS) blocking buffer (Thermo Scientific) for

30 min. In order to load single microspheres in the microwells, a microsphere stock

solution containing both anti-IL-6 Ab and anti-IL8 Ab-coated microspheres was

added to the microarray. The anti-IL-6 microspheres were encoded with 0.1 M

Eu-dye, while the anti-IL-8 Ab-coated microspheres were encoded with 0.5 M

Eu-dye. This mixture (0.5 μL aliquot) was added onto the etched end of the fiber.

The suspension was allowed to dry for 10 min and then treated with 200 μL Starting

Block Tween-20 (T20-PBS) blocking buffer (Thermo Scientific) for 30 min at RT.

For RCA-based detection of IL-6 and IL-8, the microarray was incubated in 100 μL

of Tween 20 buffer containing the varying concentrations of IL-6, IL-8, or both

(0–10 nM) for 2 h and washed. Biotinylated anti-IL-8 and anti-IL-6 detection Abs

(3 μg/mL each) were mixed together and incubated on the array for 30 min. Following

another wash, a PBS solution containing avidin (20 μg/mL) was added to the microarray for 45 min and removed. Lastly, 10 μM of biotinylated amplification primers were

added to the microarray for 45 min at RT. This allowed the conjugation of the amplification primer to the cytokine detection antibody via an avidin-biotin bridge. The construct was washed with PBS. Unless otherwise mentioned, all washing steps in this part

of the protocol were done with 1 mL of Starting Block Tween-20 (PBS) buffer.

A linear padlock probe (50 nM) was hybridized to the primer. Then an oligonucleotide “splint” (50 nM) was hybridized to the 5′ and 3′ ends of the padlock

probe for 1 h at 37 °C in the presence of ligation buffer (18.8 mM Tris–HCl,

pH 8.3, 4.6 mM MgCl2, 90.6 mM KCl, 0.15 mM NAD, 10 mM (NH4)2SO4,

3.8 mM DTT). Subsequently the padlock probe was ligated by immersing the sensor microarray in ligation buffer containing 0.1 U/μL of E. coli DNA ligase at RT

for 1 h. Finally, RCA was carried out with phi29-containing reaction buffer

(40 mM Tris–HCl (pH 7.5), 50 mM KCl, 10 mM MgCl2, 5 mM (NH4)2SO4, 4 mM

DTT with 1 U/μL Phi29 polymerase and 625 μM dNTP) for 30 min at 37 °C.

1 μM of Cy3-labeled detection probe was added for 30 min at RT to allow fluorescent

detection of RCA product.



S. Sarkar et al.

Imaging System for Microsphere Sensor Array

A customized epi-fluorescence imaging system, equipped with a mercury light source,

excitation and emission filter wheels (Chroma, Rockingham, VT), microscope objectives (Olympus, Center Valley, PA), and a CCD camera (Orca-ER, Hamamatsu), was

used to obtain all fluorescent images of the microarray. The filter wheels and shutters

were regulated by IPlab software (Scanalytics, Fairfax, VA). The fiber-optic bundle

was fixed with a chuck. The location of the microspheres on the array was assessed

using their unique fluorescent barcodes based on the concentration (0.1 or 0.5 M) of

the Eu-dye (excitation 360 nm/emission 600 nm). The RCA reaction product contained Cy3 probes, monitored using separate channels (excitation 550 nm/emission

570 nm). To maintain consistency of the fluorescence intensity, all images (encoding,

background, and signal) were acquired using 200 ms exposure time and ×20 magnification. The images were analyzed with IPlab software.



Results and Discussion

Detection of Individual RCA Amplicons on Slides

Prior to droplet experiments, we validated the sensitivity and dynamic range of our

experimental approach by using the streptavidin-coated glass slides technique

(Smolina et al. 2005) which mimics our immuno-RCA diagnostics described below.

Various amounts of complexes, composed of biotinylated primer molecules hybridized to circular DNA templates, were bound to streptavidin (Fig. 8.1). Phi29 DNA

polymerase and fluorescent (Cy3-labeled) nucleotides were added to the reaction

mixture to promote RCA at 37 °C for 4 h. Subsequent high-resolution imaging of

the slide resolved individual RCA amplicons and also yielded the total fluorescence

intensity of the scanned region (Fig. 8.1b, c). We observed a linear dynamic response

in total fluorescent intensity proportional to increasing amounts of primer-template

complexes, with the lower limit of detection corresponding to a single RCA amplicon. Such high sensitivity of detection allowed quantitative distinction of just a few

amplicons over background noise. Therefore, we hypothesized that our immunoRCA-based approach could significantly improve detection of low-abundance proteins, including the identification of single target molecules.


Amplification Protocol for Detection of Cell

Surface Proteins

We next verified the specificity and sensitivity of the immuno-RCA in biological

samples by analyzing surface protein expression in situ. Epithelial cell adhesion

molecule (EpCAM) is considered a cancer biomarker protein, since it is expressed

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