Droplet-Based Multivolume Digital Polymerase Chain Reaction by a

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Droplet-based Multi-volume Digital PCR by Surface Assisted Multifactor Fluid Segmentation Approach Wen-Wen Liu, Ying Zhu, Yi-Ming Feng, Jin Fang, and Qun Fang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03687 • Publication Date (Web): 30 Nov 2016 Downloaded from http://pubs.acs.org on December 6, 2016

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Analytical Chemistry

Droplet-based Multi-volume Digital PCR by Surface Assisted Multifactor Fluid Segmentation Approach

1 2 3 4

Wen-Wen Liu,‡ a Ying Zhu,‡a Yi-Ming Feng,b Jin Fang,b and Qun Fang*a

5 6 7 8 9

a

Institute of Analytical Chemistry, Department of Chemistry and Innovation Center for Cell

Signaling Network, Zhejiang University, Hangzhou, 310058, China b

Department of Cell Biology, China Medical University, Shenyang, 110001, China

10 11

Corresponding Author

12

*

E-mail: [email protected]. Tel.: +86-571-88206771. Fax: +86-571-88273572.

13

1

ACS Paragon Plus Environment


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Here we developed the surface assisted multifactor fluid segmentation (SAMFS), an

3

automated, fast and flexible approach for generating two dimensional droplet array with

4

tunable droplet volumes, for multi-volume digital PCR. The SAMFS was developed based on

5

the combination of robotic liquid handling and surface assisted droplet generation techniques,

6

where a continuous aqueous stream flowed out from a capillary probe was segmented and

7

immobilized on hydrophilic micropillars of a microchip into massive oil-covered droplets with

8

the probe rapidly scanning over the microchip. We studied various factors affecting the droplet

9

generation process, including micropillar top area, distance between adjacent micropillars,

10

aqueous stream flow rate and microchip moving speed, and demonstrated a high droplet

11

generation throughput up to 50 droplets/s and a largest droplet volume adjusting range from

12

0.25 nL to 350 nL. The SAMFS approach was adopted to form an oil-covered array of 994

13

droplets with four different volumes (1.2, 6, 30 and 150 nL droplets) required for multi-volume

14

digital PCR within 8 min. The droplet array system was applied in absolute quantification of

15

plasmid DNA under the multi-volume digital PCR mode with a dynamic range spanning four

16

orders of magnitude, as well as measurement of HER2 expression levels in different breast

17

cancer cell lines. The results are consistent to those obtained by quantitative real-time PCR

18

method, while the present one has higher precision.

19 20

As an emerging method for absolute gene quantification, digital PCR plays a more and more

21

important role in the field of molecular biology.1,2 In digital PCR, DNA template is randomly

22

partitioned into plenty of reactors followed by PCR reaction and then its concentration is absolutely

23

quantified by counting the number of positive reactors and calculating the results based on Poisson

24

distribution.3,4 Since the quantification is independent of amplification curve, digital PCR eliminates 2


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the uncertainties and inaccuracies from the variations in amplification efficiency, and thus it exhibits

2

higher sensitivity and accuracy compared with quantitative real-time PCR.5,6 Nowadays, digital PCR

3

has been widely used in cancer research,7,8 prenatal diagnosis,9,10 pathogen detection11,12 and

4

environmental studies.13,14

5

In recent years, microfluidic chips become the dominating platform for digital PCR, because

6

the microfluidic technique provides attractive characteristics in performing massive isolated

7

microreactions including high throughput and automated generation of massive microreactors, as

8

well as low sample/reagent consumption. Currently, various microfluidic techniques have been

9

developed to implement digital PCR, and among them three strategies have been usually employed,

10

including polydimethylsiloxane (PDMS) chip-based microchambers, SlipChip, and droplet-based

11

microfluidics using continuous flow isolation or surface-assisted method for droplet generation.

12

With PDMS chips, integrated microvalves are commonly employed to realize microchamber

13

isolation for sample partitioning.13,15 Besides microvalve, immiscible oil, such as mineral oil or

14

fluorinate oils, can also be used for compartmenting aqueous sample solution into

15

microchambers.16-18 The gas permeability of PDMS material facilitates sample self-introduction into

16

the microchambers without external equipment.19 A SlipChip20,21 device usually consists of two

17

assembled plates imprinted with wells and ducts. Sample solution is first introduced and filled the

18

connected ducts and wells, and then segmented by slipping one plate to separate wells from ducts.

19

Droplet-based microfluidics provides an economical way for high throughput microreactor isolation

20

and generation, and thus has received more and more attention in recent years, as reflected in two

21

commercial digital PCR instruments. Based on multiple-phase instability, a continuously-flowing

22

sample stream is isolated by immiscible oil into droplets in a flow focusing or T junction

23

microchannel other than chambers and wells.22,23 Commercial droplet-based digital PCR instruments, 3



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such as BioRad QX20024,25 and RainDance RainDropTM,26 can generate uniform droplets with high

2

throughput of twenty thousand droplets and ca. a million droplets per sample, respectively, and both

3

instruments are capable to perform assays for eight samples in parallel. In addition, droplet-based

4

techniques using primer-functionalized beads27,28 or agarose droplets29 instead of primer solution

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have been developed for subsequent recovery of droplet contents. Massive droplets can also be

6

generated using the surface-assisted technique30 instead of the continuous flow technique22,23. With

7

such a technique, selectively patterned hydrophilic wells or through holes are adopted to trap and

8

isolate the sample solution into a stationary, two dimensional (2D) droplet array. After droplet

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generation, fluorinated oil is usually employed to prevent droplet evaporation during PCR cycling.

10

Wide dynamic range is one of the essential considerations for a digital PCR assay, especially

11

for the applications dealing with samples with large concentration differences, such as viral load

12

evaluation31 and gene expressions in different tissue samples32. Wide assay dynamic range would

13

accelerate the measurement operation by avoiding sample serial dilution before PCR amplification,

14

and also facilitate the quantification of real-world samples with unknown concentrations. To obtain

15

wide dynamic range, most of the microfluidic digital PCR systems employed the strategy of

16

increasing the number of compartmentalized microreactors, since the dynamic range enlarges with

17

the increase of reaction number.16,22 However, simply increasing reaction number will lead to

18

significant challenge in the fabrication of high-density chambers and the detection of large numbers

19

of droplets. An alternative strategy is to use the multi-volume digital PCR method,33 where multiple

20

microreactors with different volumes are utilized. Compared with single volume digital PCR

21

systems, the multi-volume strategy can significantly reduce the total reactor number, while

22

maintaining the same dynamic range.33 For example, by theoretical calculation, 1000

23

multiple-volume reactors with volumes spanning about two orders of magnitude are predicted to 4


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offer a dynamic range similar to that of 10000 single-volume reactors. Moreover, the dynamic range

2

and resolution under the multi-volume digital PCR mode can be independently adjusted according to

3

actual requirements. Multi-volume digital PCR was first reported for quantification of HIV and

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hepatitis C virus load with the SlipChip technique.21 A wide dynamic range for each virus load

5

detection from 1.8×103 to 1.2 ×107 molecules/mL could be obtained by adopting multiple reaction

6

chambers with well volumes from 0.2 nL to 625 nL. For fabrication of multiple chambers with large

7

volume range on one glass chip, complex multi-step lithography and wet-etching procedures were

8

employed to produce wells with different depths of 30 μm and 100 μm.21 Integrated pneumatic

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micropumps were also developed for generating droplets with multiple sizes from 73 to 265 m for

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multi-volume digital PCR.34 Droplet volume was adjusted by changing the actuation pressure and

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frequency of the micropump. Recently, Xu et. al.35 described a cross-interface emulsification

12

technique, where droplets were generated by vibrating a capillary at the air-oil interface and the

13

droplet volume could be adjusted by changing sample flow rate and capillary vibrating frequency.

14

Here, we described surface assisted multifactor fluid segmentation (SAMFS), an automated and

15

rapid approach to generate 2D droplet array with multiple volumes on a microchip. The SAMFS

16

approach was developed by combining robotic liquid handling and surface assisted droplet

17

generation techniques, utilizing the advantages of the former in automated operation and accurate

18

fluid driving and control, as well as the latter’s ability in simple and reliable droplet segmentation

19

and immobilization with hydrophilic micropillar array. Massive oil-covered droplets could be

20

automatically formed with a high throughput up to 50 droplets/s and with a flexibly adjustable

21

droplet volume range up to ca. 1000 times. We applied the SAMFS approach to form droplet array

22

with four different droplet volumes from 1.2 nL to 150 nL for multi-volume digital PCR assay with a

23

dynamic range spanning four orders of magnitude. The present digital PCR system was further 5



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applied to quantify HER2 expression levels in different breast cancer cell lines to demonstrate its

2

feasibility in medical diagnosis and targeted therapy.

3 4

EXPERIMENTAL SECTION

5

Chip Fabrication. Glass chips with hydrophilic micropillar array were fabricated by using

6

photolithography and wet etching technology (see Figure 1b). Four types of micropillars with

7

different sizes (180 m, 220 m, 300 m, and 480 m diameter) and numbers (260, 294, 264, 176)

8

were designed in one microchip, to generate droplets with different volumes of 1.2 nL, 6 nL, 30 nL

9

and 150 nL, and thus obtain a wide detection dynamic range. Glass plate coated with chromium and

10

photoresist (Shaoguang, Changsha, China) was first aligned with a pre-designed photomask

11

(Kaisheng, Shanghai, China) with the micropillar array configuration and then exposed to UV light.

12

Later the glass plate was immersed in a NaOH solution (0.125 M) for development and heated at

13

110 °C for photoresist curing. Next, the exposed chromium area was removed using a chromium

14

etchant solution (HClO4/(NH4)2Ce(NO3)6, 0.6/0.365 M) to further expose the underlying glass

15

surface. After rinsed with water, the glass plate was then incubated in a wet etching solution

16

(HF/NH4F/HNO3, 1.0/0.5 /0.75 M) to etch the exposed glass area with an etching depth of 5 m. For

17

further surface modification, the patterned glass plate was first treated in 1 M NaOH solution for 20

18

min followed by drying. Then it was immersed in a n-octadecyltrichlorosilane solution (0.25 wt %,

19

Sigma, St. Louis, MO) in toluene for 30 min to perform silanization. As a result, the exposed glass

20

area became hydrophobic, while the top end area of each micropillar protected by the chromium

21

layer still maintained intrinsic hydrophilic property of glass. The residual chromium layer was then

22

totally removed using the previous chromium etchant solution. Finally, a hydrophilic micropillar

23

array surrounded with hydrophobic coating was fabricated on the glass chip. For containing cover oil, 6


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a glass frame fabricated by drilling method was glued on the micropillar array chip using epoxy

2

adhesive.

3

Instruments. The movement of microchip and liquid handling operation performed in the

4

present work were achieved using our previously-developed droplet robot platform.36 It was mainly

5

composed of a tapered capillary probe (Polytetrafluoroethylene, PTFE, 50 µm i.d., 370 µm o.d.; tip

6

size, 50 µm i.d., 160 µm o.d.)(Cole-Parmer Vernon Hills, IL), a syringe pump (PHD 2000, Harvard

7

Apparatus, Holliston, MA) with a 100 µL syringe (1710N, Hamilton, Reno, NV) and an automated

8

x-y-z translation stage (PSA series, Zolix, Beijing, China). The PTFE capillary probe, instead of

9

fused silica capillaries as in previous work37, was chosen to further eliminate sample adsorption on

10

the probe surface. The tapered tip of the capillary probe was fabricated using pulling method with a

11

hot air gun (852D, Chinafix Co., Shenzhen, China) at 270 °C.

12

In order to achieve thermal cycling and wide-field fluorescence imaging, we set up a CCD

13

camera (DH-SV1411FM, Daheng Image, Beijing, China) coupled with a Nikon camera lens (AF

14

Micro 60 mm f/2.8D, Nikon, Japan) over a commercial thermal cycler with an in-situ heating plate

15

(TC-EA/B-4IA, Bioer, Hangzhou, China). The fluorescence of the droplet array was excited at 470

16

nm by two LEDs (3W, Cree, Durham, NC) and filtered with a narrowband pass filter (535AF40,

17

Omega, Brattleboro, VT). A customized program written by LabVIEW (Version 8.0, National

18

Instruments, Austin, TX) was used for controlling shutters of the excited light and CCD camera to

19

implement in situ real time fluorescence detection. Fluorescence images captured by the CCD

20

camera were processed with background subtraction and contrast enhancement by ImageJ (NIH, MD)

21

to quantify digital PCR results.

22

Procedure Droplet generation. A schematic diagram of the setup of the present droplet-based digital PCR

23

7



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system is shown in Figure 1a. The capillary probe was connected with the syringe for fluid transport,

2

and the flow rate could be controlled precisely by the syringe pump. The movement of the microchip

3

mounted on the x-y-z translation stage was controlled using a program written by LabVIEW.

4

At first, 3 µL of mineral oil (M5904, Sigma, St. Louis, MS) and 40 µL of sample solution were

5

sequentially aspirated by the capillary probe into a PTFE storage tube (ca. 60 cm length, 300 µm i.d.,

6

760 µm o.d., Cole-Parmer, Vernon Hills, IL) connected the probe and the syringe used for

7

temporarily storing sample solution. Then, the position of the capillary probe was adjusted to a

8

distance of ca. 0.1 mm between its tip end and the microchip surface. And the mineral oil was

9

covered on the chip for preventing droplets evaporation. For droplet generation, the syringe pump

10

was set at a constant-flow-rate working mode, i.e. the aqueous sample solution continuously flowed

11

out from the tip of the capillary probe. Meanwhile, the microchip was controlled by the program to

12

move along a predetermined path, allowing the capillary probe sequentially sweep the tops of target

13

micropillars. During this process, the aqueous solution flowing out from the capillary tip was

14

adhered and restricted on the hydrophilic top area of the micropillar under the oil-layer, due to the

15

selective hydrophilic/hydrophobic surface modification to the chip. With the continuously moving of

16

the chip, the solution adhered on the micropillar top was separated from the capillary probe tip as

17

well as the solution stream flowing out from the capillary tip, forming a droplet on the micropillar

18

covered by oil. By regularly and continuously moving the chip to allow the capillary tip sweep along

19

a series of micropillars, droplet array could be generated at a high speed.

20

Digital PCR. Primers, SYBRGreen Select Master Mix (No. 4473903), Taqman Universal

21

Master Mix (No. 4440042) and TaqMan® Gene Expression Assays (hs01001580-m1 and

22

hs02758991-g1) were all purchased from Thermo Fisher Scientific Inc. (Waltham, MA). Diethyl

23

pyrocarbonate (DEPC)-treated water (TAKARA, Dalian, China) was used for sample preparation. 8


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Primer sequences for PIK3CA were as follows:

2

5’-GGAGTATTTCATGAAACAAATGAATGATGCA-3’ (forward primer)

3

5’-GAGCTTTCATTTTCTCAGTTATCTT-3’ (reverse primer).

4

For PIK3CA gene amplification, 40 µL reaction solution consisted of 20 µL of 2× SYBRGreen

5

Select Master Mix, 1.6 µL of forward primer (10 µM), 1.6 µL of reverse primer (10 µM), 0.5 µL of

6

20 mg mL-1 BSA solution (TAKARA, Dalian, China), 14.3 µL of DEPC-treated water, and 2 µL of

7

plasmid DNA (Sangon, Shanghai, China).

8

After droplet generation, the chip was degassed by vacuum and covered with a silanated

9

hydrophobic glass slide to further prevent droplet evaporation. Then the chip was placed on the

10

thermal cycler for amplification and on chip imaging. PCR thermal cycling was initiated with 4-min

11

“UNG inactivation” at 50 °C and 5-min “hot start” at 95 °C. Next, 36 cycles were performed as

12

follows: 42 s at 95 °C for denaturation, 45 s at 60 °C for primer annealing, 60 s at 72 °C for

13

extension.

14

In gene expression experiment, commercial TaqMan® Gene Expression Assays (HER2:

15

hs01001580-m1, GAPDH: hs02758991-g1) were used for target gene amplification. SKBR-3 cells

16

and MCF-7 cells (Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) in

17

suspension were collected to extract total RNA and synthesize cDNA by Sangon. The reaction

18

solution (40 µL) consisted of 20 µL of 2×Taqman Universal Master mix, 2 µL of TaqMan® Gene

19

Expression Assay (20×), 16 µL of DEPC-treated water, and 2 µL of cDNA. A two-step thermal

20

cycling protocol (3 min at 50 °C; 10 min at 95 °C; 45 cycles of [35 s at 95 °C, 70 s at 60 °C]) was

21

carried out.

22

The glass chip could be reused for more than ten times without observable performance loss.

23

Before reuse, the glass chip was treated sequentially with DNA-ExitusPlusTM (AppliChem, 9



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Darmstadt, Germany) and RNase remover (TIANDZ, Beijing, China), rinsed with deionized water,

2

and dried in a heat oven.

3 4

RESULTS AND DISCUSSION

5

System design. In this work, we aimed to develop an automated and fast approach to generate

6

droplets with multiple volumes for multi-volume digital PCR with wide dynamic range. We

7

specially intended to use a robotic system coupled with the surface-assisted technique for achieving

8

droplet generation, because such a fully automated system would provide a possibility to embed the

9

digital PCR function in the automated liquid handling platforms currently used in many biological

10

laboratories. However, the state-of-art liquid handling robots, such as Mosquito (TTP Labtech, Co.)

11

and HoneyBee (DigiLab, Inc.), have limitation in forming nanoliter-scale droplets required for

12

digital PCR assay. We have developed a droplet robot for liquid handling based on sequential

13

operation droplet array (SODA),36 which can reliably generate and manipulate droplets in the

14

picoliter to nanolitor range. It has been applied in various fields requiring nanoliter liquid handling,

15

such as enzyme reaction,36 protein crystallization,38 real-time PCR, and single cell analysis.37

16

However, the droplet generation speeds of these systems are commonly in the range of 2-10 s per

17

droplet, which are too low to be used in digital PCR. The relatively low droplet generation speeds of

18

SODA systems can be ascribed to two main reasons. First, a typical droplet generation cycle

19

includes a series of steps including x-y-z translation stage moving, stage stopping, syringe pump

20

starting, pump stopping, and stage moving again, to deposit droplet onto the target microwell. Such

21

an operation mode is suitable to handle multiple different samples in massive screening, while is

22

time consuming and low operation efficiency for generating plenty of droplets from one sample as in

23

digital PCR assay. Furthermore, the delay time between each adjacent step, as well as the low flow 10


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rates of the syringe pump for guaranteeing droplet uniformity make the operation time much longer.

2

To overcome this difficulty, we developed a surface assisted multifactor fluid segmentation

3

(SAMFS) approach to achieve automated and fast droplet generation. Differing from the previous

4

SODA technique, the SAMFS approach maintains continuous sample flow and chip/stage moving

5

without periodic pauses during the droplet generation process, i.e. the capillary probe continuously

6

scans the micropillars on the chip. The role of segmenting the continuous flow into immobilized and

7

isolated droplets is accomplished by the hydrophilic patterned micropillars. The process was

8

recorded and studied with a high-speed camera (Figure 2a, 2b, and Movie S1 in Supporting

9

Information). Figure 2a and 2b show schematic drawings and extracted images of one droplet

10

generation cycle. Aqueous sample solution is delivered out from the capillary tip driven by the

11

syringe pump at a constant flow rate (Figure 2b1). As a result of the hydrophobic property of the

12

capillary tip, the aqueous solution forms a spherical-shaped droplet on the tip, and its size increases

13

gradually (Figure 2b2). Meanwhile, the chip is moving horizontally under the control of the

14

translation stage. When the chip moves to a position where the capillary tip is just above a

15

micropillar, the aqueous droplet hung at the capillary tip adheres to the hydrophilic surface of the

16

micropillar (Figure 2b3) and a liquid bridge is formed (Figure 2b4). With the continuously moving

17

of the chip, the liquid bridge is broken up, leaving an isolated droplet immobilized on the

18

hydrophilic top area of the micropillar (Figure 2b5).

19

With the SAMFS approach, the droplet generation speed could be significantly increased by at

20

least 100 times compared with the previous SODA systems, with a highest speed of 50 droplets/s

21

(See section “Generation of multiple-volume droplets with the SAMFS approach”). In addition to

22

fast droplet generation speed, more importantly, the SAMFS approach also exhibits high flexibility

23

and reliability in controlling and changing droplet volumes by adjusting pump flow rate, and stage 11



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moving speed. Detailed study of the relationship among these parameters can be found in the next

2

section.

3

We demonstrated it can flexibly and reliably generate droplets with volumes from 0.25 nL to

4

350 nL with the same experiment setup. Figure 2c and 2d shows fluorescent images of a droplet

5

array with single volume of 10 nL and a droplet array with multi-volumes of 1.2 nL, 6 nL, 30 nL,

6

and 150 nL, respectively, using 100 M sodium fluorescein solution as a model sample.

7

Generation of multiple-volume droplets with the SAMFS approach. Since the combination

8

of the surface-assisted technique and liquid handling robot technique has not been reported for

9

oil-covered droplet generation, we investigated various factors affecting the droplet generation

10

process in details, including micropillar top surface area, distance between adjacent micropillars,

11

pump flow rate, and chip/stage moving speed.

12

In a typical surface-assisted hydrophilic spot system for droplet generation, the spot area (i.e.

13

the micropillar top area in the present system) plays an important role in droplet immobilization,

14

while is not the dominant factor to determine droplet volume. In the present system, for forming

15

large-volume droplets, larger size of micropillars is preferential to provide sufficient area to

16

immobilize and load the droplets. For forming small-volume droplets, smaller size of micropillars in

17

favor of keeping the droplet in hemispherical shape and increase its optical path in absorbance or

18

fluorescence detection. Experiments have shown that droplets with volumes ranging from 0.5 nL to

19

75 nL could be generated on a micropillar array with a diameter of 300 µm for each micropillar and

20

0.6 mm distance between adjacent micropillars by using the SAMFS approach. Besides the

21

micropillar size, an appropriate micropillar interval distance is also required to be chosen to prevent

22

droplet coalescence and meanwhile to ensure a high-density droplet array. Both the two

23

above-mentioned parameters are determined in the chip design stage prior to chip fabrication and 12


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droplet generation.

2

In addition to micropillar size and interval distance, sample flow rate and chip/stage moving

3

speed are two dominant and adjustable parameters to determine droplet volume during the process of

4

droplet generation. One major feature of the SAMFS approach is that the flow rate of the sample

5

solution flowing out from the capillary tip and the sweeping speed of the tip over the micropillar

6

array could be quantitatively controlled by the syringe pump and the stage, respectively. More

7

importantly, the sample solution flowed out from the capillary tip are totally formed into droplets on

8

the micropillars. This is different from the previous surface-assisted hydrophilic spot technique39,40

9

where only a part of sample solution was used for droplet formation, and complicated measurement

10

and calibration steps are usually required to obtain accurate droplet volumes. With such a feature, the

11

volume of each droplet could be accurately calculated by the following equation, which is equals to

12

the volume of aqueous solution flowed out from the tip during the movement from one micropillar

13

to next micropillar.

V  F T  F 

14

S



(1)

15

Where V is the droplet volume (nL), F is the flow rate of pump (nL/s), T is the time for droplet

16

generation (s), S is the distance between the adjacent micropillars (mm), and ν is the moving speed

17

of the robotic stage (mm/s). To validate the correctness of equation 1, we generated droplet arrays

18

using 50 µM sodium fluorescein solution as a model sample at different flow rate (2, 3, 4, 5, and 6

19

µL/min) and different moving speed (2, 4, 6, 8, and 10 mm/s), and measured the fluorescence

20

intensity of the droplets, respectively. Since the total fluorescence intensity of a droplet obtained

21

from its image using Image J was linearly correlated with the droplet volume in the same testing

22

range as in Figure 3 (See Figure S1), we studied the effects of sample flow rate and chip moving

23

speed on droplet generation by using the droplet fluorescence intensity as the testing parameter 13



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Page 14 of 32

1

(Figure 3a and 3b). As shown in Figure 3a and 3b, the fluorescence intensity is proportional to the

2

flow rate (R2 = 0.993), and inverse proportional to the moving speed (R2 = 0.999) with good linear

3

relationship, verifying the accuracy of equation 1. The equation 1 is also supported by the fact that

4

the calculated droplet generation time is self-consistent with the actual average value obtained by

5

recording the overall generation time of 40 droplets (see Table S1).

6

On the basis of equation 1, generation of multi-volume droplets can be conveniently realized by

7

adjusting the sample flow rate or chip moving speed instead of changing micropillar size or interval

8

distance. To further clarify the relationship of the droplet volume, sample flow rate, and chip moving

9

speed, a simulated three-dimensional plot was made at a fixed distance between adjacent

10

micropillars, in which the droplet volume was calculated using equation 1 and the variable ranges of

11

the parameters were acquired from actual experiments (Figure 3c). Taking a micropillar array with

12

300 µm micropillar size and 0.6 mm interval distance as an example, 0.5-nL droplets can be

13

generated at a flow rate of 0.75 µL/min and a moving speed of 15 mm/s. By increasing the flow rate

14

to 6 µL/min meanwhile slowing the moving speed to 0.8 mm/s, 75 nL of droplets can be generated

15

in the same micropillar array, achieving 150-fold variation in droplet volume.

16

For the multi-volume droplet array shown in Figure 2d, two different flow rate (1.44 µL/min

17

and 9.00 µL/min) and four different moving speed (6.0, 1.6, 3.0, and 0.9 mm/s) were employed to

18

generate droplets with volumes of 1.2 nL, 6 nL, 30 nL and 150 nL, respectively, where the

19

coefficients of variation of droplet volumes are in the range of 3.9% to 7.8 %. Actually, by flexibly

20

adjusting the combination of the flow rate and moving speed, a larger droplet volume variation range

21

from 0.25 nL to 350 nL could be conveniently generated with the same micropillar array chip.

22

It should be noted that the SAMFS approach has intrinsic difference from the

23

previously-reported surface-assisted technique. In the well and through-hole based surface-assisted 14


Page 15 of 32

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1

techniques, droplet volume is mostly decided by the well volumes.30,41 Adjusting reaction volumes

2

requires redesigning and fabrication of new microchips. With well-free surface-assisted

3

technique,39,40 droplets are formed on the surface of hydrophilic spots. The droplet volumes are

4

decided by multiple factors such as spot surface area, surface tension of aqueous sample solution,

5

and sliding speed during droplet generation process, which is required to be measured and calibrated

6

before the assay.

7

Performance of multi-volume digital PCR. For realizing multi-volume digital PCR with wide

8

dynamic range, we generated a droplet array consisted of four different volume droplets (260 1.2-nL,

9

294 6-nL, 264 30-nL, and 176 150-nL droplets) in 8 min. The volumes were increased by a

10

multiplication factor of five, for reaching a compromising balance between the quantification

11

resolution and dynamic range. With this multi-volume design, the system provides a dynamic range

12

from 83 to 3.7 × 106 copies/mL, spanning 4.6 orders of magnitude.

13

To test the performance of the multi-volume digital PCR system, we performed digital PCR

14

assay using a serial of dilutions of synthetic PIK3CA plasmid DNA with final concentrations ranging

15

from 1.1×102 copies/mL to 1.1×106 copies/mL. For each concentration, the experiment was tested 3

16

times. With the in-situ real time fluorescence detector, fluorescent images of multi-volume droplet

17

array at each cycle were automatically captured, which are help to distinguish the noise and

18

amplified signals.22 Figure 4a shows the end-point fluorescent images of negative control and

19

samples of plasmid DNA with five different concentrations spanning four-order of magnitudes. As

20

the DNA concentration increases, more positive droplets are observed. According to the principle of

21

multi-volume digital PCR,33 for the samples with DNA template concentrations below 104

22

copies/mL, large volume droplets (150 nL) contributes most to the final calculated results. With the

23

DNA template concentration increases, medium volume droplets (30 nL and 6 nL) make the major 15



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Page 16 of 32

1

contribution in the result calculation. And small volume droplets (1.2 nL) acts an important role for

2

the calculation of concentration exceed 106 copies/mL.

3

By counting the number of positive droplets in each set of individual volumes, the DNA

4

template concentrations were calculated using a Matlab program33 based on Poisson distribution. As

5

shown in Figure 4b, the calculated concentration scales linearly with the expected concentration, and

6

the correlation coefficient between the two sets of values is 0.9997, which indicates the reliability of

7

the present multi-volume digital PCR system. We also plotted the ratio of calculated/expected

8

concentrations against the expected concentration (Figure 4c). In 15 experiments, 12 fell within the

9

95% confidence interval and all experiments fell within the 99% confidence interval. An analysis

10

between concentrations calculated by statistical analysis of all droplets with different volumes and

11

that calculated from each set of data of droplets with individual volumes, shows a self-consistent

12

result, further indicating the reliability of the present system for gene quantification (see Figure S2).

13

Measurement of HER2 gene expression. To demonstrate the potential of the present

14

multi-volume digital PCR system in medical diagnosis, we applied it to study HER2 gene expression

15

in different breast cancer cells. The HER2 gene expression level is closely related to patient

16

prognosis in breast cancer. High levels indicate high-risks of tumor invasion and metastasis, and

17

imply targeted therapy may be employed for patients. Two different types of breast cancer cells,

18

SKBR-3 and MCF-7 cells, were chosen as the model samples, for the reason that HER2 gene is

19

highly expressed in SKBR-3 cells while poorly expressed in MCF-7 cells. In order to demonstrate

20

the feasibility and performance of the present digital PCR assay method, the RNA extraction,

21

purification and reverse transcription for the samples were carried out using routine method15,19,32

22

instead of in-droplet method to reduce the uncertainty of experimental results. We prepared the

23

cDNA solution by reverse transcription from total RNA extracted from each culture cell line. Then 16


Page 17 of 32

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1

we employed the present system to absolutely quantify HER2 target gene and GAPDH reference

2

gene, respectively. The housekeeping GAPDH gene was used to normalize different input cDNA

3

amounts due to the variation of input cell numbers. Figure 5a illustrates the results of multi-volume

4

digital PCR in the quantification of HER2 (blue cycles) and GAPDH (blue squares) expression

5

levels in cDNA from SKBR-3 and MCF-7 cells, respectively. Significant different expressions of

6

HER2 gene in the two types of cells could be observed, which is in concordance with the previously

7

reported results 42. We next compared the result with that obtained using conventional quantitative

8

real-time PCR method. As shown in Figure 5b, the ratio of HER2 gene expression between SKBR-3

9

and MCF-7 cells was measured to be 5.15 ± 0.09 (n=3) by digital PCR, and 5.01 ± 0.77 (n=3) by

10

quantitative real-time PCR, indicating the better precision of the multi-volume digital PCR method

11

over the quantitative real-time PCR method.

12 13

CONCLUSIONS

14

In summary, we have developed an automated multi-volume digital PCR system with the

15

SAMFS approach, which realizes rapid generation of 2D droplet arrays with multiple volume

16

droplets directly under oil layer. This system was successfully applied in the absolute quantification

17

of nucleic acid and gene expression analysis of cDNA samples reverse-transcribed from different

18

breast cancer cells with good repeatability and wide dynamic range. The present work provides a

19

promising solution for coupling liquid handling robot technique with digital PCR assay. It also

20

provides an effective way for rapidly forming large scale of 2D droplet array covered by oil.

21

Differing from other microfluidic techniques with fixed volume of droplets or microchambers,

22

the volume of droplets formed by the SAMFS approach is mainly determined by the flow rate of

23

pump and moving speed of robotic stage, which can be easily and flexibly adjusted. In comparison 17



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Page 18 of 32

1

with the SlipChip system using fixed-volume chambers to conveniently form droplets with different

2

sizes, the SAMFS approach is able to change droplet volume without the need of redesigning a new

3

chip. This offers convenience in adjusting the detection range of digital PCR assay according to the

4

actual requirements of different samples using the same chip. Since the present digital PCR assay

5

system was built on the basis of an automated robotic platform, it also has the potential in coupling

6

automated sample pretreatment with digital PCR assay. Meanwhile, due to the semi-open

7

characteristic of the present droplet array chip, it could be applied in subsequent gene analysis of

8

amplification products by employing the capillary to sample from target droplets through the cover

9

oil, like secondary PCR and gene sequencing. In addition, the SAMFS approach could also be

10

incorporated with a wide range of applications, including isothermal amplification, DNA library

11

preparation prior to sequencing, and single cell analysis, etc.

12

The SAMFS approach could be further developed in the future. To further improve the droplet

13

generation throughput, microchip with higher micropillar density or multiple capillary probes could

14

be used. The multiple capillary system coupled with multiple chips could also be used to implement

15

simultaneous multi-sample PCR assays. With smaller inner diameter of capillary or faster chip

16

moving speed, droplet volumes could be further decreased to picoliter scale. If some treatments,

17

such as primer bonding, protein bonding or chemical modification, are carried out to the micropillars

18

before droplet generation, sample pretreatment function may also be integrated with the system.

19 20 21

ACKNOWLEDGMENT ‡

Wen-Wen Liu and Ying Zhu contributed equally to this work.

22

Financial supports from Natural Science Foundation of China (Grants 21475117, 21435004, and

23

81327004), and Natural Science Foundation of Zhejiang Province (Grant LY14B050001) are 18


Page 19 of 32

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1


gratefully acknowledged.

2 3

SUPPORTING INFORMATION

4

This material is available free of charge via the Internet at http://pubs.acs.org.

5

19



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Page 20 of 32

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1 2

FIGURE CAPTIONS

3

Figure 1. (a) Schematic diagram of the micropillar array chip. The insets show an enlarge image of

4

the droplet during the generation process and a photograph of a micropillar array chip with different

5

micropillar sizes. (b) Schematic diagram of fabrication process of a chip with hydrophilic

6

micropillar array.

7

Figure 2. Schematic drawings (a1-a5) and images (b1-b5) of a droplet generation cycle. Scale bar,

8

200 μm. Fluorescent images of a 38 × 38 droplet array with uniform droplet volume of 10 nL (c) and

9

a multi-volume droplet array with 1.2 nL, 6 nL, 30 nL, and 150 nLdroplet volumes (d).

10

Figure 3. (a) Linear correlation between droplet fluorescence intensity and sample flow rate at a

11

fixed chip moving speed of 5 mm/s. (b) Inversely proportional relationship between droplet

12

fluorescence intensity and reciprocal of chip moving speed at a fixed sample flow rate of 3 μL/min.

13

(c) Simulated three-dimensional plot showing the relationship of droplet volume, sample flow rate,

14

and chip moving speed at a fixed distance between adjacent micropillars.

15

Figure 4. Performance of the present multi-volume digital PCR. (a) End-point fluorescent images of

16

multi-volume droplet arrays in digital PCR assays with a serial dilution of plasmid DNA and

17

negative control. (b) Comparison of the expected concentrations of plasmid DNA to the calculated

18

concentrations. (c) Comparison of the expected concentrations of plasmid DNA to the ratios of

19

calculated/expected concentration.

20

Figure 5. (a) HER2 gene expression levels in MCF-7 and SKBR-3 cells by multi-volume digital

21

PCR. The normalized ratio of HER2 to GAPDH in gene expression is represented by the red

22

diamonds. (b) Ratios of HER2 gene expression between SKBR-3 and MCF-7 cells measured by the

23

present multi-volume digital PCR and conventional quantitative real-time PCR, respectively.

24 25



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Page 26 of 32

1 2

(a)

Capillary

Micropillar Oil

Droplet

Micropillar

Frame Micropillar array chip

(b) Mask Cr Glass Photoresist

UV light

Exposure

Cr Removal

Wet Etching Hydrophilic surface Hydrophobic surface

AZ Removal

Silanization

3 4

Figure 1

5 6 7

26


Cr Removal

Page 27 of 32

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1 2

(a1)

Capillary

Micropillar

(b1) Micropillar

Capillary

Droplet

Oil 4

3

Chip

2

1

0.00 s

(a2)

Droplet

(b2)

4

3

2

1

0.03 s

(a3)

(b3)

4

3

2

0.07 s

(a4)

(b4)

Liquid bridge 4

3

2

0.11 s

(a5)

(b5)

4

3

2

0.13 s

(d)

(c)

1 mm

2 mm

3 4 5

Figure 2

6

2 mm

7 27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2

(a)

R2 = 0.993

(b) R2 = 0.999

(c)

3 4 5

Figure 3

6 7 8 9

28


Page 28 of 32

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1 2 3

(a)

Negative control

1.1×102 copies/mL

1.1×103 copies/mL

1.1×104 copies/mL

1.1×105 copies/mL

1.1×106 copies/mL

(b)

(c)

R2=0.9997

4 5 6

Figure 4

7 8

29



1 2

Concentration (copies/μL)

(a)

MCF-7 cells

SKBR-3 cells

(b)

dPCR

qPCR

4 5 6

Figure 5

7 8

30


Normalized HER2 : GAPDH ratio

3

Ratio (SKBR-3 : MCF-7)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 Droplet array

Capillary

Micropillar Droplet

Oil

2 mm

Micropillar

Frame Micropillar array chip

3 4

For TOC only

5 6 7 8

31


1 mm


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Droplet-Based Multivolume Digital Polymerase Chain Reaction by a

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