Inkjet printing based droplet generation for integrated on-line digital PCR

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Inkjet printing based droplet generation for integrated on-line digital PCR Weifei Zhang, Nan Li, Daisuke Koga, Yong Zhang, Hulie Zeng, Hizuru Nakajima, Jin-Ming Lin, and Katsumi Uchiyama Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00463 • Publication Date (Web): 29 Mar 2018 Downloaded from http://pubs.acs.org on March 29, 2018

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Analytical Chemistry

Inkjet printing based droplet generation for integrated on-line digital PCR †‡

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Weifei Zhang, Nan Li, Daisuke Koga, Yong Zhang, Hulie Zeng, Hizuru Nakajima, Jin-Ming Lin,‡* and Katsumi Uchiyama,†*

†Department of Applied Chemistry, Graduate School of Urban Environmental Sciences, Tokyo Metropolitan University, Minamiohsawa, Hachioji, Tokyo 192-0397, Japan. Fax: +81-426-77-2821. E-mail: [email protected] ‡Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China. Email: [email protected]

ABSTRACT: We report on the development of a novel and flexible on-line digital PCR (dPCR) system. The system was composed of three parts: an inkjet for generating the droplets, a coiled fused-silica capillary for thermal cycling and a laser induced fluorescence detector (LIFD) for positive droplet counting. Upon inkjet printing, monodisperse droplets were continuously generated in the oil phase and then introduced into the capillary in the form of a stable dispersion. The droplets containing one or zero molecules of target DNA passed through the helical capillary that was attached to a cylindrical thermal cycler for PCR amplification, resulting in the generation of fluorescence for the DNA-positive droplet. After 36 PCR cycles, the fluorescence signal intensity was detected by laser-induced fluorescence located at the downstream of the capillary, followed by a positive/negative counting. The present system was successfully applied to the absolute quantification of the HPV sequence in Caski cells with dynamic ranges spanning four orders of magnitude.

The occurrence of a disease generally leads to changes in overall cytopathy, eventually resulting in tissue necrosis. If these lesions could be detected at the singlemolecule level at an early stage, this could increase the survival period of patients, even allowing them to recover. Therefore, the development of a method of detection at the single-molecule level would have great significance for basic biomedical research, medical diagnostics and drug discovery. However, it should be noted that the levels of such molecules in cells is quite low, which would require their amplification prior to analysis and detection. The polymerase chain reaction (PCR) has become the most significant tool for single nucleic acid analysis in modern biology and medical applications, owing to its dramatic amplification capability, which permits many copies of a molecule to be made from a single sequence. Digital PCR (dPCR) has become a promising alternative for the detection of single copies of nucleic acids, due to its ability for achieving absolute quantification. Using this technique, targets are distributed into numerous microreactors, thus permitting the number of positive signals to be counted. 1-4

The progress of digital PCR is closely related to the development of partition techniques, such as, dividing the reaction mixture into a large number of micro tubes or chambers in microfluidic devices;2,5-9 generating numerous water-in-oil (W/O) emulsion droplets as microreactors.1,10-13 Emulsion-based methods for producing droplets for dPCR are the preferred methodology, because the oil water interface permits every droplet as an individual micro-reactor with an adjustable volume range from the pico-liter to the nano-liter scale in a short time. Although, the microfluidic device-based droplet-generating technique is quite popular and is in common use, it should be noted that the high cost and the complex fabrication continues to be a problem in terms of application. After that, the droplets have to be transferred to detecting machines to count the positive ones. Inevitably, this multi-step procedure causes some droplets to be lost or infected. Therefore, new approaches for dPCR that would be simple, reproducible and economical would be highly desirable. Printing technology, especially inkjet printing, represents an alternate method for producing mono-disperses micro-droplet for use in digital PCR assays, since the

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technique has the advantages of both high throughput and permits the precise control of small volumes.14-18 This technique, which can be precisely manipulated and dispersed with spatial and temporal control, does not require a complicated fabricating process and is now employed in many areas, thus making it one of the most promising methods for efficient microarray fabrication.19 In addition, droplet volume can also be controlled through adjusting the driving voltage and applied pulse waveform on the piezo actuator of the inkjet system.20-23 Yang et al. reported that inkjet technology can be used for the preparation of monodisperse polymer particles.24 Sun and co-worker reported on a new sequential-inkjet printing model for generating pico-liter muti-component droplet-in-oil arrays on uniform planar substrates.25 To the best of our knowledge, generating monodisperse droplets with an inkjet system for use in digital PCR has not been reported. In this study, we report on the development of an integrated and flexible system for performing digital PCR. The system is composed of three parts: droplet generation using inkjet printing methodology, a coiled capillary for thermal cycling, and a laser induced fluorescence detector (LIFD) for detection. Using inkjet printing, monodisperse droplet can be accurately generated and stably dispersed in a continuous oil phase, significantly simplifying the emulsification process and reducing reagent consumption. The generating droplets continuously pass through a cylindrical thermal-cycling system at different temperatures 36 times for DNA amplification by PCR. Finally, the positive droplet that contains a target DNA is counted by the LIFD. By virtue of this on line dPCR system, we were able to achieve on-line amplification and detection without any liquid transfer steps, thus avoiding crosscontamination and sample loss. We applied this system to the quantitative analysis of various concentrations of DNA ranging across three orders of magnitude and the findings demonstrate its feasibility for use in medical diagnosis and related applications. Experimental section Device Fabrication. The on-line dPCR device (Figure 1) consists of three parts: generating droplets by inkjet printing, a coiled capillary for thermal cycling, and an LIFD system for detecting fluorescence signals. The inkjet system was used to produce monodisperse droplets with different sizes by controlling the driving condition of a piezo actuator on an inkjet microchip (pulse voltage and pulse width). A VW-9000 high-speed microscope was employed to observe the droplet ejected by the inkjet (Kyence Corporation, Osaka, Japan). The aqueous droplets (50~110 um in diameter) were entrained by an immiscible oil phase and introduced into the fused silica capillary (530-μm-i.d., GL Science, Tokyo, Japan). The carrier fluid used in all experiments was mineral oil (SIGMA, Tokyo, Japan) and the mineral oil was supplemented with 2 wt% of a Span® 80 surfactant (SIGMA, Tokyo, Japan). The 4.5 m long capillary was helically folded 36 times around a cylindrical thermal-cycling unit. (Figure S1).

Three temperature controlled copper blocks were used for denaturing, annealing and extension for the PCR process. Each copper block was mounted on each side of the plastic core, forming an assembly with a cylindrical appearance (30 mm in diameter). Adjacent copper blocks have an air gap of ~4 mm for thermal insulation. The thin film heater (80 mm x 30 mm; China, UXCELL) and a temperature sensor (2 mm in diameter and 27 mm in length; China, SODIAL) are mounted into each copper block, so that the temperature of each block can be controlled independently by means of a temperature regulator (50 oC ~ 110 oC; SODIAL). For the experiments discussed here, we used a denaturing temperature of 94 oC, an annealing temperature of 55 oC, and an extension temperature of 72 o C. A vacuum pump (ASONE, Japan) installed on the end of the capillary was used to control the flow rate of the oil phase. The time for each cycle is determined by the diameter of the temperature regulating equipment and the flow rate of the oil phase that carry droplets. The PCR amplification product is directly detected in the reaction capillary by laser-induced fluorescence detection (LIF) at the end of the capillary. A solid blue laser (HK-5517-02, 473 nm, Shimadzu, Kyoto, Japan) is used as an excitation light source and incident into the capillary through a 10 x microscope objective lens (Olympus, Tokyo, Japan). The fluorescence signal was collected through the same objective lens and filter set (U-MW1B2 Olympus, Tokyo, Japan) and the signal was then sent to a photomultiplier tube (PMT) (H6780-02, Hamamatsu photonics, Shizuoka, Japan). The fluorescence signal was recorded using a CDS plus ver 5.0 (Lasoft. Co.,.Tokyo, Japan.). DNA Extraction. DNA was extracted from Caski cells (Cancer Insitute and Hospital, Chinese Academy of Medical Science, Beijing, China) using a TIANamp Micro DNA Kit (TIANGEN, China, Beijing) according to the manufacturer's recommended protocol. The DNA concentration was estimated from the A260/A280 absorption using a U3900s spectrophotometer (Hitachi, Japan). All samples were stored at -20 oC. Droplet Digital PCR Reaction. The dPCR reaction mixtures were prepared in 1.5 mL centrifuge tubes as follows. 20 μL PCR mixture solution containing 10 μL SYBR Premix Ex TaqⅡ (TAKARA Bio inc.), 0.4 μL ROX reference dye, 0.8 μL each of primers (forward: 5’- 3’ GCACAG GGACATAATAATGG; reverse: 5’ – 3’ CGTCCCAAAGGA AACTGATC), 2 μL of template DNA and 6 μL double distilled water (ddH2O). Before amplification, the mixture was heated at 95 oC for 3 min to activate enzymes. The mixture was then transferred to the inkjet sampling tube and then subjected to thermal cycling through our designed dPCR system for DNA amplification and detection. The whole process cycled 36 times with 94 oC for 10 s, 55 o C for 20 s and 72 oC for 20 s. Treatments of Capillary Inner Surface. In order to maintain stable droplets and avoid adsorption on the inner surface of a fused-silica capillary, surface coating steps were used to make the inner surface of capillary hydro-


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Analytical Chemistry phobic. At first, a new fused-silica capillary was rinsed with 0.1 M NaOH, 0.1 M HCl and deionized water for 15 min each to activate the capillary. Then, AquapelTM (Pittsburgh Glass Works, LLC, Pittsburgh, PA) solution was injected into the capillary for 10 min to make inner surfaces hydrophobic, followed by drying with air.

We optimized the inkjet conditions and selected six different sizes of droplets, produced by the above procedure for the following experiment (50, 65, 75, 90, 100 and 110 µm). As shown in Figure 3, the generated droplets had a uniform size distribution and their size could be controlled by adjusting driving waveform. The uniformity of droplet size was evaluated according to coefficient of variation (CV) of the droplets, which is defined as CV = (σ/μ x 100) %, where σ represents the standard deviation and μ represents the average diameter. The CV values from all experiments were below 4.41 %. Such good monodispersity was quite significant for the uniform distribution of the target DNA and uniform delivery for the progress of amplification. The findings reported herein demonstrate that an inkjet printing system could be used to reliably generate droplets with droplet sizes from 50 to 110 µm. Figure S2 shows the fluorescent intensity of

Figure 1. Schematic diagram of the digital PCR system based on inkjet technology.

Results and discussion Generation of monodisperse droplets. The operating principle of the system is shown schematically in Figure 1. An inkjet printing system was used to generate highly uniform monodisperse droplets containing PCR reaction solutions. Theoretically, the PCR solution was diluted until there was no more than one target DNA molecule in the droplet. As shown in Figure 2, the inkjet nozzle was immersed into the oil phase, generating droplets with volumes of pico-liters to nano-liters, the size of which could be adjusted by controlling the inkjet system conditions (driving voltage and pulse width). The speed of droplet-generation could also be regulated to permit a high throughput to be achieved by changing the frequency of the applied pulse. After droplet generation followed by their introduction into the capillary, the droplets were thermal-cycled in the coiled capillary tube, which was mounted onto the three different temperature control modules. Each droplet, which was separated by the continuous oil phase, can be considered to be an individual reaction unit of PCR. Details of the process can be found in the supplementary Video S1, which was recorded by a high speed camera. The size of droplets was closely related to the characteristics of the fluid and the waveform applied to the piezo actuator on the inkjet. As shown in Table S1, monodisperse droplets could be reproducibly produced when the pulse width was fixed at 10 us, and a driving voltage ranging from 30 to 100 V was used. On the other hand, when the driving voltage was fixed at 20 V, the pulse width that could successfully eject droplets was in the range of 20 990 us. To avoid damage to the inkjet by the high voltage and large pulse width, the maximum driving voltage and pulse width were set below 100 V and 990 us, respectively.

Figure 2. Principle of monodisperse droplet generation by the inkjet system. Typical photograph of monodisperse droplets jetting into a continuous oil phase. The four different color of the inkjet represent four channels. We use the second channel for PCR mixed reagent loading.

Figure 3. Size distribution of droplets generated by the inkjet under various conditions for both driving voltage and pulse width.



Figure 4. Influence of injection frequency on the resolution of the fluorescence signal between two droplets. (a-f) Frequencies for droplet injection were a: 1 Hz, b: 0.66 Hz, c: 0.5 Hz, d: 0.4 Hz, e: 0.33 Hz f: 0.28 Hz, Test sample (100 μM sodium fluorescein solution), and pressure of the pump; 45 kPa, Capillary coil: without heating.

different-sized droplets (50 ~ 110 µm), and a 100 µM sodium Fluorescein solution was used as a test sample. The signal intensity was enhanced with increasing droplet size. In comparison with the conventional microfluidic device- based droplet-generating technique, this inkjetbased droplet-generating method was much easier to operate and the size of the ejected droplets could be controlled more easily. Optimization of droplet ejection frequency. The inkjet printing technique has the capability for producing large numbers of droplets within a short time. In order to realize high throughput droplets using the on-line PCR system, a stable liquid introduction system, an on-line thermal cycling system, and a high throughput fluorescence detection system are necessary and they need to be effectively mated. In order to transport the droplets through the capillary tube, a high-performance vacuum pump located at the end of the capillary was used to remove the oil phase by aspiration. Such a high-throughput droplet-generation system can also be combined with a high speed fluorescence detection system for counting droplets contain the target DNA. However, it should be noted that high frequency droplet generation reduces the corresponding distance between two droplets, leading to the droplet fusion or junction a in the process of PCR amplification. To address this issue, we further investigated the influence of ejection frequency on droplet fusion. Figure 4 shows fluorescence signal patterns for droplets generated at various ejection frequencies ranging from 1 to 0.28 Hz. As shown in Figure 4a and 4b, some inconsecutive strong peaks can be seen, indicating that two or more droplets had fused to form a larger droplet or contact was made between adjacent droplets. As shown in Figure 4c ~

4f, the signal intensities among the adjacent droplets were almost uniform and repetitive concomitant with the decrease in ejection frequency. When the injection frequency reached 0.33 Hz, the droplet fusion was practically not observed, and the droplets were individually in the capillary. The results suggested that an appropriate distance between droplets was of great importance for achieving reproducible fluorescence detection. The droplets were ejected at a frequency of 0.33 Hz in the following experiments unless otherwise specified. Velocity of droplet transport. The stable flowthrough PCR system was not only beneficial for the detection system, but also played a crucial role in the PCR reaction itself. The principle of a flow reactor is an attractive concept for application to PCR. The three static temperature zones eliminated the need for temperature cycling and minimized associated thermal hysteresis. Thus, the technology offered the potential for extremely fast DNA amplification. However, some constraints also need to be taken into consideration. The temperature of the reaction mixture did not change immediately from one region to another new temperature level, and some time was required to finish this progress. This time was dependent on the flow and thermal conductivity of the transfer tube and the reaction liquid. Thus, the use of a fused-silica capillary as transfer tube would be advantageous due to its superior thermal conductivity. Low viscosity mineral oil (35 cp) with 2 wt% Span 80 was used as a continuous phase. The oil-soluble surfactants Span 80 could effectively reduce the surface tension of the oil phase and increase the stability of the droplets during the reaction process.


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Analytical Chemistry

Figure 5. The relationship between pump pressure and the velocity of the monodisperse droplet PCR cycle.

The flow rate of the oil phase to carry monodisperse droplets in the continuous phase is one of the most important parameters in determining PCR reaction efficien-

cy. The flow-through dPCR reaction system was constructed so as to have 36 identical PCR cycles. The time for each cycle was evenly divided for 1) denaturing, 2) annealing, and 3) extension. These reaction times were determined by the flow rate of a monodisperse droplet through the capillary. Figure 5 shows the relation between pump pressure and droplet velocity flowing through the 36 cycles capillary. Droplet velocity was increased with increasing pump pressure in the range of 0.155 ~ 0.6 cm/s. Generally speaking, an amplification time of one cycle is about 50-60 s and the length of one cycle in our heating equipment is 10 cm. Please note that the temperature of the flow phase in the capillary is approximated by the temperature of the capillary wall. Besides, the droplet had large specific surface area, thus heat transfer efficiency can be improved. At the same time, the amplification efficiency of the droplet could be guaranteed due to the high heat transfer efficiency. Given these, we used an optimized velocity of 0.21 cm/s, a pump of pressure 10 Kpa in the following dPCR experiment. The time for the entire process was relatively smaller than the traditional PCR

Figure 6. Performance of the present digital PCR system. (a) Fluorescence signal of droplets for the serial dilution of DNA. The conditions for inkjet ejections were a 100 V driving voltage and a 10 μs pulse width for which the size of all droplets was controlled to 100 μm. (b) Fluorescence signal of droplets for serially diluted DNA and negative control. (The fluorescence signal shown in (a), (b) are the partial peak from 1-9 minutes).



amplification time. Real sample analysis. In order to confirm the feasibility and performance of the present dPCR system, an actual sample was analyzed. The DNA extracted from Caski cells were diluted into a series (100, 101, 102, 103 of dilution ratios). For each corresponding concentration, 3, parallel experiments were conducted. Figure 6a shows the endpoint fluorescent signal for various dilutions of DNA samples after PCR amplification (The conditions for the inkjet were 100 V driving voltage and 10 μs pulse width, the size of all droplets was controlled at 100 μm). As can be seen, it was possible to calculate the sample concentration when the sample was diluted 102 times according to the principle of dPCR and the Poisson distribution. However, when the sample was diluted at ratios of 100 or 101, almost all of the droplets contained one or more target molecules. When the sample was diluted to magnification of 103, very few droplets contained target molecules owing to the low sample concentration. As a result, we optimized the droplet size for different sample dilutions. As shown in Figure 6b, when the samples contained the DNA template at a concentration below 102 dilution, large size droplets (100 um and 110 um) played an important role in the final calculated results. When the DNA template concentration was increased, medium size droplets (75 um to 50 um) made the major contributor to the calculated results. The data were processed using Excel, the signal intensity of negative signal was regarded as noise, all of the numerical values for intensity exceeded the noise by three times was assumed to be a positive signal. Thus, noise can be distinguished from amplified signals based on signal intensity. The number of positive signals increased with increasing DNA concentration. The concentration of the DNA template was calculated based on Poisson distribution via counting the number of positive droplets showing a stronger signal intensity in each set, as shown in Table S2. As shown in Figure 7, there is a perfect linear relationship between the calculated concentrations and serial diluted concentrations that were prepared, i.e., y = - 0.978 x + 4.380. The correlation coefficient R2 between the two sets of values was 0.9996, confirming the

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feasibility of the present dPCR system. The reproducibility of the experimental results is very good in this integrated on-line system, which has no need to manually transfer the droplets for amplification and detection. Importantly, this system also avoids the cross-contamination during the transfer process. Conclusion In summary, we report on the development of an automated on-line digital PCR system based on an inkjet technique, which permitted the convenient generation of monodisperse droplets and their direct injection into an oil phase, followed by an effective amplification of DNA inside the droplet during their passage through a coiled capillary tube. This inkjet generator greatly simplified the experimental setup and operation procedure with low cost and low reagent consumption, and the size of the monodispersed droplets could be precisely regulated by the inkjet actuator. We successfully performed dPCR for the absolute quantification of the HPV sequence in Caski cells. In comparison with previous reports, our system was stable, flexible and inexpensive, without liquid transfer steps eliminated material loss or cross-contamination. We believe that this home-made equipment has the potential to be widely applied in diverse areas, including biological and chemical research.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Information on PCR thermal-cycling system, correlated ejection conditions for generating droplets, fluorescein intensities of different-sized droplets, Count the positive signal and calculate the copies/μL in each set samples.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. *E-mail: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by High technology research funding from Tokyo Metropolitan University, National Key R&D Program of China (2017YFC0906800) and the National Natural Science Foundation of China (No. 21435002, 21621003).

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Figure 7. The correlation between the measured concentration and the dilution ratio of target DNA templates.

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Inkjet printing based droplet generation for integrated on-line digital PCR

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