A temperature controlled microfluidic system ... - ACS Publications

Subscriber access provided by Iowa State University | Library

Article

A temperature controlled microfluidic system incorporating polymer tubes Jiu Yang Zhu, Ngan Nguyen, Sara Baratchi, Peter Thurgood, Kamran Ghorbani, and Khashayar Khoshmanesh Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05365 • Publication Date (Web): 28 Dec 2018 Downloaded from http://pubs.acs.org on January 1, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 23 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

Analytical Chemistry

A temperature controlled microfluidic system incorporating polymer tubes

Jiu Yang Zhu 1,*, Ngan Nguyen 1,*, Sara Baratchi 2, Peter Thurgood 1, Kamran Ghorbani 1, Khashayar Khoshmanesh 1,†

1 2

School of Engineering, RMIT University, Melbourne, Victoria 3001, Australia

School of Health and Biomedical Sciences, RMIT University, Bundoora, Victoria 3083, Australia

* These authors have contributed equally † Corresponding author: [email protected]

1 ACS Paragon Plus Environment

Analytical Chemistry 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

Abstract Here, we demonstrate a multi-layered microfluidic system integrated with commercially available polymer tubes for controlling the temperature of the sample under various static and dynamic conditions. Highly controllable temperature profiles can be produced by modulating the flow rate or inlet temperature of the water passing through the tubes. Customised temperature gradients can be created across the length or width of a channel by mismatching the inlet temperature of the tubes. Temperature cycles can also be produced by repeatedly switching the tubes between hot and cold flasks. Proof-of-concept experiments demonstrate the utility of this system for studying the drug-induced calcium signalling of human monocytes under dynamic thermal conditions. The versatility and simplicity of our system provides opportunities for studying temperature-sensitive chemical, biochemical and biological samples under various operating conditions.

Keywords: Microfluidics, lab-on-a-chip, integrated, self-sufficient, temperature control


Page 2 of 23

Page 3 of 23 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. Introduction Innovative microfluidic systems enable a wide range of cellular and molecular assays using small amounts of biological samples and reagents

1-5.

Temperature is an environmental

parameter that regulates many physical, chemical, and biological processes

6-9.

The majority

of current microfluidic systems take advantage of off-chip technologies such as conventional cell culture incubators

10, 11,

temperature controlled stages

miniaturised incubators 14

12,

microscope incubators

13,

and

for temperature control of the sample. Incorporation of

heating/cooling elements into the microfluidic systems reduces the overall size and cost of the experimental setup, and facilitates the development of highly integrated, self-sufficient devices 15. Commercially available indium tin oxide coated glass slides 16, 17 facilitate uniform heating of the microfluidic structure via the Joule effect. In comparison, metallic micro-heaters patterned onto the glass slides facilitate localised heating of the system 18, 19. For microfluidic systems made of soft elastomers such as PDMS (polydimethylsiloxane), miniaturised resistive heaters

20-23,

microwave generators

24

or Peltiers

25, 26

can be easily embedded in

PDMS during the fabrication process. Customised heaters can also be made by patterning conductive materials such as conductive epoxy 27 or emerging liquid metal alloys 28. An alternative solution is to pattern heat carrying auxiliary channels within the PDMS block to recirculate hot or cold liquid. The heat transfer through the auxiliary channels is governed by forced convection, which can be easily modulated by varying the flow rate or temperature of the liquid. The liquid temperature can be adjusted by implementing external heating/cooling elements

29, 30,

exothermic/endothermic reactions occurring inside the

auxiliary channels 31, as well as Joule heating for the case of ionic solutions 32. The auxiliary channels can be patterned close to the main channel to enable the rapid exchange of heat between the neighbouring channels 31, 32. This integrated system can be fabricated in a single step if the main and auxiliary channels have the same height. Despite simple fabrication process, the risk of liquid leakage between the neighbouring channels, and the free convective heat loss via the glass slide can limit the proficiency of this design. These limitations can be addressed by fabricating multi-layered microfluidic structures, realised by multi-step lithography 33, injection moulding 34 or 3D printing 35, 36 methods. In this case, the auxiliary channels are patterned close to the main channel while far enough from the glass slide to reduce the heat loss caused by free convection. Despite these advantages, the rather complex and time-consuming processes associated with the fabrication of such devices might 3 ACS Paragon Plus Environment


limit their widespread application. Innovative fabrication methods, which can be implemented using simple steps, and preferably do not rely on cleanroom facilities, can accelerate the growth of emerging of microfluidic technologies 2, 37, 38. In this work, we introduce a simple process for fabrication of multi-layered microfluidic structures integrated with commercially available polymer tubes for temperature regulation of the sample. The tubes are embedded in PDMS close to the microfluidic channel in arbitrary ‘parallel’ or ‘cross’ configurations. Varying the temperature or flow rate of the water passing through the tubes allows for creating customised temperature profiles within the channel without the risk of leakage. The tubes are isolated, and therefore can be filled with hot and cold water independently. This facilitates the creation of customised temperature gradients across the length or width of the microfluidic channel. The temperature of the channel can be dynamically modulated by applying alternating hot and cold flows through the tubes. This capability is utilised for oscillating the temperature in repeated cycles, which is used for studying the intracellular calcium signalling of human monocytes in response to low concentrations of drugs. Overall, our system can be easily fabricated and operated. The position and configuration of the tubes can be tailored according to user needs for various applications.


Page 4 of 23

Page 5 of 23 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


2. Materials and Methods 2.1 Principles of the system The experimental setup is illustrated in Figure 1. It comprises of a microfluidic system to facilitate the flow of desired samples with embedded Tygon® tubes (ID = 0.5 mm, OD = 1.5 mm, Sigma-Aldrich) to facilitate the convective heating/cooling of the sample. The hot/cold water is stored in an insulated flask and is withdrawn through the Tygon® tubes using a syringe pump (Harvard PHD ULTRA 4400). The temperature of the water inside the flask is monitored using a K-type thermocouple (TC Measurement & Control Pty. Ltd.). The sample is applied to the microfluidic channel using a secondary syringe pump (Harvard Pico Plus) and is collected in the waste bottle. An infrared camera (FLIR systems, Thermo Vision A320, Sweden) interfaced with ThermaCAM researcher software is used to monitor the temperature along the microfluidic channel in real time (Figure 1a). The Tygon® tubes are arranged in ‘parallel’ and ‘cross’ configurations. In parallel configuration, the two Tygon® tubes are placed at either side and in parallel with the microfluidic channel (Figure 1b). The gap between the tubes (centre-to-centre) is set to 5 mm to reduce the conductive loss via the PDMS block. In cross configuration, the Tygon® tubes are placed normal to the microfluidic channel (Figure 1c). The gap between the tubes is set to 10 mm to facilitate the creation of locally hot/cold regions along the middle section of the microfluidic channel bounded between the two tubes. Under steady-state conditions, the heat balance of the system can be described using Equation (1), in which QTube is the amount of heat injected into the system via Tygon® tubes, QChannel is the amount of heat effectively transferred to the microfluidic channel, and QLoss is the heat loss across the free surfaces of the PDMS block, glass coverslip and Tygon® tubes: 𝑄𝑇𝑢𝑏𝑒 = 𝑄𝐶ℎ𝑎𝑛𝑛𝑒𝑙 + 𝑄𝐿𝑜𝑠𝑠

(1)

The first two terms are governed by forced convection mechanism and can be described by 𝑄 = 𝑚 𝑐𝑝 ∆𝑇 (𝑚 is the mass rate, 𝑐𝑝 is the specific heat capacity of the liquid and ∆𝑇 is the

temperature gradient), whereas the last term is governed by free convection mechanism and can be described by ℎ 𝐴 (𝑇𝑆 ― 𝑇∞) (h is the free convective coefficient, A is the surface area, TS is the surface temperature and T∞ is the ambient temperature).



Figure 1. Experimental setup: (a) Schematics of experimental setup consisting of a microfluidic structure with embedded Tygon® tubes for convective heating/cooling of the sample, a syringe pump for infusing the sample through the microfluidic channel, a syringe pump for pulling hot/cold water through the Tygon® tubes, a water flask for storing the hot/cold water, a bottle for collection of the waste sample, an infrared camera interfaced with an analysis software for real-time monitoring of temperature across the glass coverslip. (b-c) Microfluidic structures with embedded Tygon® tubes in ‘parallel’ and ‘cross’ configurations.


Page 6 of 23

Page 7 of 23 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


2.2 Fabrication process The microfluidic system is fabricated in five major steps, as explained below: Master fabrication: A master mould with dimensions of 60×1×0.25 mm (L×W×H) was fabricated from photoactive resin using stereolithography technique with a bench-top 3D printer (Form 2, Formlabs, USA) (Figure 2a). Pouring of PDMS first layer: PDMS base and curing agents (Sylgard® 184 Silicone Elastomer Kit) were mixed in 10 to 1 weight ratio and degassed. The master was placed in a standard 85 mm glass Petri dish, and the PDMS mixture was poured onto the master to a total depth of 1 mm (Figure 2b). The first PDMS layer was kept thin to minimise the conductive loss between the Tygon® tubes and the microfluidic channel. Patterning of tubes: Once this thin PDMS layer was fully cured, two Tygon® tubes were placed on top of the PDMS surface in the desired ‘parallel’ or ‘cross’ configurations (Figure 2c). To ensure the tubes remain taut and straight, each end of the tubes was clamped to the Petri dish using small alligator clamps. Pouring of PDMS secondary layer: A second layer of PDMS was then poured onto the Tygon® tubes, resulting in a total PDMS height of 5 mm (Figure 2d). The secondary PDMS layer was kept thick to minimise the heat loss from the top surface of the PDMS block via free convection. Assembly: The cured PDMS block was peeled off the master, and plasma bonded (Harrick Plasma, Basic Plasma Cleaner) onto a glass coverslip with dimensions of 75×25×0.13 mm (L×W×H) (Bellco Brand) (Figure 2e). This thin coverslip minimised the heat conduction and facilitated the monitoring of temperature via the infrared camera. The utilisation of Tygon® tubes simplified the fabrication process, avoided leakage of liquid from the tubes even at high flow rates, and facilitated customised heating and cooling processes by adjusting the temperature of the water applied to the tubes. The experimental microfluidic system with integrated Tygon® tubes is shown in Figure S1.



Figure 2. Schematics of fabrication process: (a) A master mould was 3D printed. (b) The first layer of PDMS was poured onto the master mould. (c) Two Tygon® tubes were placed onto the top surface of the cured PDMS. (d) The second layer of PDMS was poured to cover the Tygon® tubes. (e) The PDMS block with embedded microfluidic channel and Tygon® tubes was peeled off the master and assembled onto a glass coverslip.


Page 8 of 23

Page 9 of 23 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


2.3 Numerical simulations Numerical simulations were performed to further understand the thermal characteristics of the microfluidic system. The simulations were conducted using ANSYS Fluent software (ANSYS Inc., Canonsburg, USA). This involved solving the differential equations governing the balance of mass, momentum, and energy balance inside the microfluidic channel and Tygon® tubes along with solving the energy balance differential equation inside the PDMS block, glass coverslip, and Tygon® tube walls, as detailed in Supporting Information S2.

2.4 Cellular assays Human monocytic THP-1 cells (TIB-202, ATCC) were cultured in complete RPMI 1640 medium containing 10% fetal bovine serum (Sigma) in 5% CO2 at 37°C. Changes in the intracellular calcium level [Ca2+]i of cells was measured 39, 40. Briefly, cells were immersed in standard Hanks’ Balanced Salt Solution (HBSS) buffer and loaded with Fluo-4-AM (Sigma). The cells were then seeded onto the poly-l-lysine coated glass coverslips overnight. Coverslips were mounted to the microfluidic channel using a mechanical clamp to avoid leakage. Cultured cells were excited with a 488 nm laser using Nikon A1 laser scanning confocal microscope (Nikon Instruments Inc.). Fluorescence emission was detected using a photomultiplier tube and a 525/50 nm band-pass filter using a 4× objective. Calcium influx was measured as an increase in the fluorescent intensity of Fluo-4-AM and normalised to the fluorescent intensity of resting cells. Image analysis was performed using Nikon NISElements imaging software.



3. Results and Discussions 3.1 Thermal characterisation of the system A comprehensive set of experimental and numerical analyses was conducted to characterise the thermal performance of the system. To do so, we characterised the ‘parallel’ tube configuration system when operating under reference conditions, defined as Qsample = 60 µL/min, Qwater = 500 µL/min and Twater = 58 °C. The temperature contours were captured at the surface of the coverslip, and at least 30 minutes after the operation of the system to ensure steady-state conditions were reached. Measurements indicated the formation of a hot region (coloured in yellow) along the path of the tubes (Figure 3a). Simulations revealed the formation of a hot region at the core of the PDMS block surrounding the Tygon® tubes, and relatively large temperature gradients (~17 °C) across the width of the PDMS block due to the low thermal conductivity of PDMS (Figure 3b). Analysis of temperature distribution along the microfluidic channel indicated the formation of a ‘hot region’ with a temperature of 37 ± 0.5 °C at the middle regions of the channel spanning across ~70% of its length (Figure 3c). Simulations also indicated a temperature drop of ~8 °C along the Tygon® tubes (Figure S2). The overall heat consumption of the system was calculated as 𝑄Tube = 0.48 W, 12% of which was transferred to the microfluidic channel while the rest was lost via the free convection across the external surfaces of the system, as described in Equation (1). Extended simulations indicated that increasing the gap between the Tygon® tubes raises the free convective loss and consequently the overall heat consumption (Figure S3). Next, we characterised the thermal performance of the ‘cross’ tube configuration system under reference conditions, defined as Qsample = 60 µL/min, Qwater = 500 µL/min and Twater = 56 °C (Figure 3aʹ). Both experimental and numerical results indicated the formation of a ‘hot region’ at the middle regions of the channel bounded between the two Tygon® tubes (Figure 3bʹ). The temperature contours were dilated toward the outlet of the channel due to the dominance of forced convection inside the microfluidic channel. Analysis of temperature distribution along the microfluidic channel revealed the extent of the ‘hot region’ (Figure 3cʹ). Simulations indicated a temperature drop of ~5.8 °C along the Tygon® tubes (Figure S2). The overall heat consumption of the systems was calculated as 𝑄Tube = 0.34 W, 17% of which was transferred to the microfluidic channel. Increasing the gap between the Tygon®


Page 10 of 23

Page 11 of 23 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


tubes increased the overall heat consumption of the system similar to the ‘parallel’ tube configuration (Figure S3). We conducted additional experiments to characterise the thermal performance of the microfluidic system under various operating conditions, as presented in Figures S4-S10. Extended numerical simulations indicated the capability of our system for avoiding unwanted temperature rise of the sample due to Joule heating effect (Figures S11 and S12), suggesting its potential applications in electrokinetically driven microfluidic devices 41, 42.

Figure 3. Characterising the thermal performance of the microfluidic system under reference conditions, defined as Qwater = 500 µL/min, Qsample = 60 µL/min, Twater @ parallel = 58 °C, Twater @ cross = 56 °C: (a-aʹ) Temperature contours at the surface of the glass coverslip obtained by infrared camera. (b-bʹ) Temperature contours obtained by numerical simulations. (c-cʹ) Variations of temperature along the microfluidic channel obtained by infrared camera and numerical simulations, indicating the extent of ‘hot region’.



Page 12 of 23

3.2 Spatial modulation of temperature We conducted further experiments to investigate the capability of our system for creating customised temperature gradients along the microfluidic channel. This was facilitated by inserting the two Tygon® tubes in separate flasks containing hot and cold water. Experiments were

performed

for

‘parallel’

and

‘cross’

tube

configurations

by

applying

Qwater = 500 µL/min, Thot water = 65 °C, and Tcold water = 17 °C while varying Qsample from 0 to 60 µL/min. Figure 4a presents the temperature contours for the ‘parallel’ tube configuration when applying Qsample = 60 µL/min. The results indicated the formation of hot (red) and cold (blue) regions along the PDMS block, creating a temperature gradient with a maximum of ∆𝑇𝑚𝑎𝑥 ~3.7 °C across the width of the channel (Figure 4b). Reduction of Qsample increased the temperature gradient across the channel due to dominance of conductive heat transfer (Figure 4b-inset + Figure S13). Figure 4aʹ presents the temperature contours for the ‘cross’ tube configuration when applying Qsample = 60 µL/min. The results indicated a temperature gradient of ∆𝑇𝑚𝑎𝑥~10 °C along the length of the channel (Figure 4bʹ). Reduction of Qsample increased the temperature gradient along the channel (Figures S14). Our experiments showcased the versatility of our integrated microfluidic system for creating customised temperature gradients along the width or length of the channel by simply mismatching the temperature of water flowing through the two Tygon® tubes.


Page 13 of 23 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


Figure 4. Inducing spatial temperature gradients across the microfluidic channel when applying hot and cold waters through the Tygon® tubes: (a-a′) Temperature contours at the surface of the glass coverslip obtained by infrared camera when applying Qwater = 500 µL/min, Qsample = 60 µL/min, Thot

water

= 65°C, Tcold

water

= 17°C, (b) Variations of

temperature across the width of the microfluidic channel with the ∆𝑇𝑚𝑎𝑥 representing the highest temperature gradient. Inset shows the variations of ∆𝑇𝑚𝑎𝑥 against Qsample. (b′) Variations of temperature along the length of the microfluidic channel when varying Qsample from 0 to 60 µL/min.



Page 14 of 23

3.3 Dynamic modulation of temperature We also investigated the capability of our microfluidic system for oscillating temperature between 32 and 37 °C, providing opportunities for studying thermal stress on cells

6, 13.

This

was achieved by using two flasks containing hot and cold waters. The temperature along the microfluidic channel was monitored in real-time using the infrared camera. The Tygon® tubes were transferred from the hot flask to the cold flask once the temperature at the specified location of the channel reached 37 °C and vice versa. Experiments were conducted for both ‘parallel’ and ‘cross’ tube configurations, and under Qwater = 500 µL/min, Thot water = 65 °C, and Tcold water = 14°C, Qsample = 60 µL/min conditions. Dynamic temperature contours were captured at the surface of the coverslip for the ‘parallel’ tube configuration (Movie S1). The temperature contours were extracted and analysed in 5 s intervals during the cooling (from 0 to 45 s) and heating (from 45 to 115 s) phases (Figures 5a-b). Results indicated dynamic temperature changes along the entire length of the microfluidic channel with the highest changes occurring close to the channel inlet (Figures 5c-d). In this manner, we were able to oscillate the temperature of Point A (located 20 mm from the inlet) between 32 and 37°C over successive cycles (Figure 5e). Next, we investigated the dynamic temperature modulation for the ‘cross’ tube configuration (Movie S2 + Figure 5aʹ-bʹ). Results indicated dynamic temperature changes at the region bounded between the two Tygon® tubes (Figures 5cʹ-dʹ), enabling us to oscillate the temperature of Point Aʹ (located 34 mm from the inlet) between 32 and 37°C over successive cycles (Figure 5eʹ). Our results indicated that the ‘heating phase’ was longer than the ‘cooling phase’ for both ‘parallel’ and ‘cross’ tube configurations. For the ‘parallel’ tube configuration the ratio of ‘heating phase/cooling phase’ duration was 1.55, which increased to 2.3 for the ‘cross’ tube configuration. To explain this behaviour, we assumed that the temperature of the PDMS block is constant such that the dynamic response time of the system could be estimated as follows: ∂𝑇

𝑚 𝑐𝑝 ∂𝑡

|𝑃𝐷𝑀𝑆 = 𝑄𝑛𝑒𝑡

(2)

During the ‘cooling phase’, the system loses heat both through the Tygon® tubes and its free surfaces via natural convection and 𝑄net cooling = | ― 𝑄Tube ― 𝑄Loss|. In comparison, during the ‘heating phase’, the system gains heat through the Tygon® tubes while loses heat via its free 14 ACS Paragon Plus Environment

Page 15 of 23 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


surfaces and 𝑄net heating = |𝑄Tube ― 𝑄Loss|. Given that 𝑄net cooling > 𝑄net heating the system responded faster during the ‘cooling phase’. Our extended experiments indicated that the dynamic response of the system can be shortened by increasing Qwater. For example, for the ‘parallel’ tube configuration, increasing Qwater from 500 to 1000 µL/min shortened the temperature cycle from 115 to 70 s.

Figure 5. Analysing the thermal performance of the microfluidic channel under dynamic conditions when varying temperature between 32 and 37 °C: (a-aʹ) Temperature contours obtained by infrared camera during successive cooling and (b-bʹ) heating phases. (c-cʹ) Variations of temperature along the microfluidic channel at various time steps corresponding to successive cooling and (d-dʹ) heating phases. (e-eʹ) Dynamic temperature variations at Points A and Aʹ. 15 ACS Paragon Plus Environment


3.4 Studying the drug-induced calcium signalling of cells Next, we investigated the capability of our temperature-controlled microfluidic system for inducing the drug-induced intracellular calcium signalling of human monocytes when dynamically increasing the medium temperature from 24 to 37°C (room temperature to body’s physiological temperature). To do so, the cells were seeded onto a coverslip, as detailed in Section 2.4. The microfluidic system with ‘cross’ tube configuration was chosen due to its ability for providing a localised hot region across the middle of the channel located between the two tubes, regardless of the temperature and flow rate of the liquids flowing through the channel or Tygon® tubes (Figure S4aʹ-dʹ). The microfluidic system was assembled onto a cell-coated coverslip (Figure 6a) and sealed using a mechanical clamp to avoid leakage (Figure S15). The cell culture medium containing 10 µM Yoda-1 (a selective agonist for Piezo-1 calcium permeable ion channel) was applied into the microfluidic channel at 60 µL/min. The increase in intracellular calcium level ([Ca2+]i) of cells was measured in real-time using a confocal microscope, as explained in Section 2.4. First, we conducted a control (static) experiment by placing the cell-cultured microfluidic system in a temperature-controlled microscope incubator (Clear State Solution Pty Ltd., Australia). The control experiments revealed the maximum fold increase of [Ca2+]i as 1.03 ± 0.02 at 24 °C which increased to 1.40 ± 0.41 at 37 °C. Next, we studied the [Ca2+]i of cultured cells when dynamically increasing the medium temperature from 24 to 37°C using our integrated microfluidic system (Movie S3). Temperature contours were captured at the surface of the coverslip over a period of 10 minutes (Figure 6b). Cell imaging was conducted across a rectangular region of 2000 µm × 500 µm (L×W) at the middle of the microfluidic channel that located between the two Tygon® tubes (Figure 6a). The temperature was almost uniform across the imaging region, with a maximum standard deviation of 0.27°C measured across this region during the transient conditions. The cell imaging region reached a stable temperature of 37 °C within ~8 minutes (Figure 6c). Microscopic analysis revealed the immediate increase of intracellular calcium signalling of cells in response to temperature increase (Figure 6d + Movie S4). Figure 6e presents the normalised [Ca2+]i of 15 selected cells analysed in realtime with the average [Ca2+]i shown as a thick red line. The maximum fold increase of [Ca2+]i measured by the microfluidic system was obtained as 1.41 ± 0.72, which was in good 16 ACS Paragon Plus Environment

Page 16 of 23

Page 17 of 23 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


agreement with the results of the control experiments using the microscope incubator. The cells reached their peak response within 10.7 minutes, approximately 2.7 minutes after the medium temperature became stable at 37°C (Figure 6e-inset). Our extended calculations revealed that [Ca2 + ]𝑖 ∝ 𝑇0.729 𝑖𝑚𝑎𝑔𝑖𝑛𝑔 𝑟𝑒𝑔𝑖𝑜𝑛.



Figure 6. Studying the intracellular calcium signalling of human monocytes under druginduced shear stress when increasing the temperature of the cell culture medium from 23 to 37 °C: (a) Schematics of experimental setup using the ‘cross’ tube configuration. (b) Temperature contours at the surface of the coverslip obtained by the infrared camera. (c) Dynamic variations of temperature along the cell imaging region. (d) Calcium signalling of cells in response to 1 µM Yoda-1 agonist. (e) Normalised fluorescent intensity profiles for 15 selected cells. The average response of cells is shown with a thick red line. Inset shows the increase of [Ca2+]i and Qsample over time.


Page 18 of 23

Page 19 of 23 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


Conclusion In summary, we demonstrated an integrated microfluidic system capable of heating and cooling of the sample under both static and dynamic conditions. The system takes advantage of commercially available Tygon® tubes embedded in PDMS, which are patterned close to a microfluidic channel. The tubes are patterned in ‘parallel’ and ‘cross’ configurations with respect to the channel. Highly controllable and predictable temperature profiles were created by varying the inlet temperature or flow rate of the water passing through the tubes. Various temperature gradients were produced by mismatching the inlet temperature of the tubes. Furthermore, we showed the ability to oscillate the temperature between 32 and 37°C in successive cycles by alternating the hot and cold flows through the tubes. The system was utilised for studying the drug-induced calcium signalling of human monocytes when increasing the temperature from 24 to 37°C. This suggests the potential applications of the system for various cellular experiments, and in particular for studying the response of thermo-sensitive cells. Customised temperature profiles can be provided by tailoring the configuration of Tygon® tubes. In particular, the ‘parallel’ tube configuration provides an almost uniform temperature profile along the entire length of the microfluidic channel, whereas the ‘cross’ tube configuration provides a localised temperature profile along the regions of the channel bounded between the two tubes. Future work includes the integration of commercially available miniaturised pumps (i.e. piezoelectric pumps), and temperature sensors into the current system to enable a self-sufficient, portable microfluidic system

15, 43,

and application of highly conductive liquid metal alloys through the heat carrying Tygon® tubes to further enhance the convective heat transfer 44.

Supporting Information: The Supporting Information is available free of charge on the ACS Publications website. SI 1: Image of microfluidic systems with integrated Tygon® tubes in ‘parallel’ and ‘cross’ configurations, SI 2: Details of numerical simulations, SI 3: Temperature drop along the Tygon® tubes for the ‘parallel and ‘cross’ tube configurations, SI 4: Investigating the effect of the gap between the Tygon® tubes on the thermal performance of the system, SI 5: Thermal characterisation of the microfluidic system under various operating conditions, SI 6: Thermal characterisation of the microfluidic system when Joule heating effect occurs along the microfluidic channel, SI 7: Thermal characterisation of the microfluidic system when 19 ACS Paragon Plus Environment


applying hot and cold water through the Tygon® tubes, SI 8: Utilisation of a mechanical clamp to avoid leakage during cellular assays. Conflict of interest: Authors declare no conflict of interest. Acknowledgements: S. B. acknowledges the Australian Research Council for Discovery Award for Early Career Researchers (DE170100239). K. K. acknowledges the Australian Research Council for Discovery Grants (DP180102049 and DP170102138). References 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Zhang, W.; Zhang, Y. S.; Bakht, S. M.; Aleman, J.; Shin, S. R.; Yue, K.; Sica, M.; Ribas, J.; Duchamp, M.; Ju, J.; Sadeghian, R. B.; Kim, D.; Dokmeci, M. R.; Atala, A.; Khademhosseini, A., Elastomeric free-form blood vessels for interconnecting organs on chip systems. Lab Chip 2016, 16 (9), 1579-1586. N. Nguyen; P. Thurgood; J. Y. Zhu; E. Pirogova; S. Baratchi; Khoshmanesh, K., “Do-itin-classroom” fabrication of microfluidic systems by replica moulding of pasta structures. Biomicrofluidics 2018, 12 (4), 044115. C. Hajal; M. Campisi; C. Mattu; V. Chiono; Kamm, R. D., In vitro models of molecular and nano-particle transport across the blood-brain barrier. Biomicrofluidics 2018, 12 (4), 042213. Hosic, S. J.; Murthy, S. K.; Koppes, A. N., Microfluidic Sample Preparation for Single Cell Analysis. Anal. Chem. 2016, 88 (1), 354-380. Zhang, D. X.; Bi, H. Y.; Liu, B. H.; Qao, L., Detection of Pathogenic Microorganisms by Microfluidics Based Analytical Methods. Anal. Chem. 2018, 90 (9), 5512-5520. Miralles, V.; Huerre, A.; Malloggi, F.; Jullien, M.-C., A Review of Heating and Temperature Control in Microfluidic Systems: Techniques and Applications. Diagnostics 2013, 3 (1). Chon, C. H.; Li, D., Temperature Control in Microfluidic Systems. In Encycl. Microfluid. Nanofluid., Li, D., Ed. Springer US: Boston, MA, 2008; pp 1976-1980. Kim, S. C.; Clark, I. C.; Shahi, P.; Abate, A. R., Single-Cell RT-PCR in Microfluidic Droplets with Integrated Chemical Lysis. Anal. Chem. 2018, 90 (2), 1273-1279. Goertz, J. P.; White, I. M., Phase-Change Partitions for Thermal Automation of Multistep Reactions. Anal. Chem. 2018, 90 (6), 3708-3713. McCormick, S.; Tong, Z.; Ivask, A.; Morozesk, M.; Voelcker, N. H.; Lombi, E.; Priest, C., Optimization of binding B-lymphocytes in a microfluidic channel: surface modification, stasis time and shear response. Biofabrication 2017, 10 (1), 014101. Yang, J.; Zheng, M.; Yang, F.; Zhang, X.; Yin, W.; Liu, X.; Zhang, G.-J.; Chen, Z., Pseudomonas aeruginosa infected nematode-on-a-chip model array for antibacterials screening. Sens. Actuators, B 2018, 275, 373-381. Gouriou, Y.; Bijlenga, P.; Demaurex, N., Mitochondrial Ca(2+) Uptake from Plasma Membrane Cav3.2 Protein Channels Contributes to Ischemic Toxicity in PC12 Cells. J. Biol. Chem. 2013, 288 (18), 12459-12468. Baratchi, S.; Tovar-Lopez, F. J.; Khoshmanesh, K.; Grace, M. S.; Darby, W.; Almazi, J.; Mitchell, A.; McIntyre, P., Examination of the role of transient receptor potential vanilloid type 4 in endothelial responses to shear forces. Biomicrofluidics 2014, 8 (4), 044117. 20 ACS Paragon Plus Environment

Page 20 of 23

Page 21 of 23 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


14. Weng, L.; Ellett, F.; Edd, J.; Wong, K. H. K.; Uygun, K.; Irimia, D.; Stott, S. L.; Toner, M., A highly-occupied, single-cell trapping microarray for determination of cell membrane permeability. Lab Chip 2017, 17 (23), 4077-4088. 15. Boyd-Moss, M.; Baratchi, S.; Di Venere, M.; Khoshmanesh, K., Self-contained microfluidic systems: a review. Lab Chip 2016, 16 (17), 3177-3192. 16. Williams, S. J.; Chamarthy, P.; Wereley, S. T., Comparison of Experiments and Simulation of Joule Heating in ac Electrokinetic Chips. J. Fluids Eng. 2010, 132 (2), 021103-021103-7. 17. Silva Santisteban, T.; Rabajania, O.; Kalinina, I.; Robinson, S.; Meier, M., Rapid spheroid clearing on a microfluidic chip. Lab Chip 2018, 18 (1), 153-161. 18. Bridle, H.; Millingen, M.; Jesorka, A., On-chip fabrication to add temperature control to a microfluidic solution exchange system. Lab Chip 2008, 8 (3), 480-483. 19. Fang, C.; Lee, D.; Stober, B.; Fuller, G. G.; Shen, A. Q., Integrated microfluidic platform for instantaneous flow and localized temperature control. RSC Adv. 2015, 5 (104), 85620-85629. 20. Fu, R.; Xu, B.; Li, D., Study of the temperature field in microchannels of a PDMS chip with embedded local heater using temperature-dependent fluorescent dye. Int. J. Therm. Sci. 2006, 45 (9), 841-847. 21. Yi, P.; Awang, R.; S T Rowe, W.; Kalantar-zadeh, K.; Khoshmanesh, K., PDMS nanocomposites for heat transfer enhancement in microfluidic platforms. Lab Chip 2014, 14. 22. Pardy, T.; Tulp, I.; Kremer, C.; Rang, T.; Stewart, R., Integrated self-regulating resistive heating for isothermal nucleic acid amplification tests (NAAT) in Lab-on-a-Chip (LoC) devices. PLOS ONE 2017, 12 (12), e0189968. 23. Wu, J.; Cao, W.; Wen, W.; Chang, D. C.; Sheng, P., Polydimethylsiloxane microfluidic chip with integrated microheater and thermal sensor. Biomicrofluidics 2009, 3 (1), 012005. 24. Issadore, D.; Humphry, K. J.; Brown, K. A.; Sandberg, L.; Weitz, D. A.; Westervelt, R. M., Microwave dielectric heating of drops in microfluidic devices. Lab Chip 2009, 9 (12), 1701-1706. 25. Maltezos, G.; Johnston, M.; Scherer, A., Thermal management in microfluidics using micro-Peltier junctions. Appl. Phys. Lett. 2005, 87 (15), 1-3. 26. Rosengarten, G.; Mutzenich, S.; Kalantar-zadeh, K., Integrated microthermoelectric cooler for microfluidic channels. Exp. Therm Fluid Sci. 2006, 30 (8), 821-828. 27. Vigolo, D.; Rusconi, R.; Piazza, R.; Stone, H. A., A portable device for temperature control along microchannels. Lab Chip 2010, 10 (6), 795-798. 28. Tang, S.-Y.; Lin, Y.; Joshipura, I. D.; Khoshmanesh, K.; Dickey, M. D., Steering liquid metal flow in microchannels using low voltages. Lab Chip 2015, 15 (19), 3905-3911. 29. Velve-Casquillas, G.; Costa, J.; Carlier-Grynkorn, F.; Mayeux, A.; Tran, P. T., A Fast Microfluidic Temperature Control Device for Studying Microtubule Dynamics in Fission Yeast. Methods Cell Biol. 2010, 97, 185-201. 30. Chung, K.; Crane, M. M.; Lu, H., Automated on-chip rapid microscopy, phenotyping and sorting of C. elegans. Nat. Methods 2008, 5, 637. 31. Guijt, R. M.; Dodge, A.; van Dedem, G. W. K.; de Rooij, N. F.; Verpoorte, E., Chemical and physical processes for integrated temperature control in microfluidic devices. Lab Chip 2003, 3 (1), 1-4. 32. de Mello, A. J.; Habgood, M.; Lancaster, N. L.; Welton, T.; Wootton, R. C. R., Precise temperature control in microfluidic devices using Joule heating of ionic liquids. Lab Chip 2004, 4 (5), 417-419.



33. Unger, M. A.; Chou, H.-P.; Thorsen, T.; Scherer, A.; Quake, S. R., Monolithic Microfabricated Valves and Pumps by Multilayer Soft Lithography. Science 2000, 288 (5463), 113-116. 34. Li, X.; Soler, M.; Szydzik, C.; Khoshmanesh, K.; Schmidt, J.; Coukos, G.; Mitchell, A.; Altug, H., Label‐Free Optofluidic Nanobiosensor Enables Real‐Time Analysis of Single‐Cell Cytokine Secretion. Small 2018, 14 (26), 1800698. 35. Shallan, A. I.; Smejkal, P.; Corban, M.; Guijt, R. M.; Breadmore, M. C., Cost-Effective Three-Dimensional Printing of Visibly Transparent Microchips within Minutes. Anal. Chem. 2014, 86 (6), 3124-3130. 36. Lee, Y.-S.; Bhattacharjee, N.; Folch, A., 3D-printed Quake-style microvalves and micropumps. Lab Chip 2018, 18 (8), 1207-1214. 37. Trantidou, T.; Regoutz, A.; Voon, X. N.; Payne, D. J.; Ces, O., A “cleanroom-free” and scalable manufacturing technology for the microfluidic generation of lipid-stabilized droplets and cell-sized multisomes. Sens. Actuators, B 2018, 267, 34-41. 38. Nguyen, D.; McLane, J.; Lew, V.; Pegan, J.; Khine, M., Shrink-film microfluidic education modules: Complete devices within minutes. Biomicrofluidics 2011, 5 (2), 022209. 39. Baratchi, S.; Almazi, J. G.; Darby, W.; Tovar-Lopez, F. J.; Mitchell, A.; McIntyre, P., Shear stress mediates exocytosis of functional TRPV4 channels in endothelial cells. Cell. Mol. Life Sci. 2016, 73 (3), 649-66. 40. Baratchi, S.; Knoerzer, M.; Khoshmanesh, K.; Mitchell, A.; McIntyre, P., Shear Stress Regulates TRPV4 Channel Clustering and Translocation from Adherens Junctions to the Basal Membrane. Sci. Rep. 2017, 7 (1), 15942. 41. Mata-Gomez, M. A.; Perez-Gonzalez, V. H.; Gallo-Villanueva, R. C.; Gonzalez-Valdez, J.; Rito-Palomares, M.; Martinez-Chapa, S. O., Modelling of electrokinetic phenomena for capture of PEGylated ribonuclease A in a microdevice with insulating structures. Biomicrofluidics 2016, 10 (3), 033106-033106. 42. Xuan, X., Joule heating in electrokinetic flow. Electrophoresis 2008, 29 (1), 33-43. 43. Thurgood, P.; Zhu, J. Y.; Nguyen, N.; Nahavandi, S.; Jex, A. R.; Pirogova, E.; Baratchi, S.; Khoshmanesh, K., A self-sufficient pressure pump using latex balloons for microfluidic applications. Lab Chip 2018, 18, 2730-2740 44. Meng, G.; Lin, G., Development of a fast thermal response microfluidic system using liquid metal. J. Micromech. Microeng. 2016, 26 (7), 075005.


Page 22 of 23

Page 23 of 23 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


for TOC only


A temperature controlled microfluidic system ... - ACS Publications

Recommend Documents