Polydimethylsiloxane Microchannel Research Articles

Recent Advances of PDMS In Vitro Biomodels for Flow Visualizations and Measurements: From Macro to Nanoscale Applications

Polydimethylsiloxane (PDMS) has become a popular material in microfluidic and macroscale in vitro models due to its elastomeric properties and versatility. PDMS-based biomodels are widely used in blood flow studies, offering a platform for improving flow models and validating numerical simulations. This review highlights recent advances in bioflow studies conducted using both PDMS microfluidic devices and macroscale biomodels, particularly in replicating physiological environments. PDMS microchannels are used in studies of blood cell deformation under confined conditions, demonstrating the potential to distinguish between healthy and diseased cells. PDMS also plays a critical role in fabricating arterial models from real medical images, including pathological conditions such as aneurysms. Cutting-edge applications, such as nanofluid hemodynamic studies and nanoparticle drug delivery in organ-on-a-chip platforms, represent the latest developments in PDMS research. In addition to these applications, this review critically discusses PDMS properties, fabrication methods, and its expanding role in micro- and nanoscale flow studies.

Parallel Concentric Wrinkled Polydimethylsiloxane Patterns and Their Potential Biomedical Applications

AbstractThe objective of this study is to examine microbial behavior by fabricating and applying parallel concentric wrinkled patterns to polydimethylsiloxane (PDMS) substrates. The patterns are generated and subsequently relaxed in a rigid silica layer, resulting in the formation of discernible wrinkles that can be analyzed using electron microscopy and light diffraction. The alignment of E. coli and silica microparticles is facilitated by these structures along the creases. Within these creases, E. coli exhibits enhanced attachment and rotational mobility in regions characterized by hydrophobic cracks. Furthermore, the bacteria demonstrate an increase in velocity when propelled by the PDMS creases, suggesting that these structures are capable of guiding microbial motion. In addition, distinct alignment and growth patterns are observed for E. coli proliferation within the PDMS microchannels under confined conditions. The analysis of bacterial morphology and gene expression in response to chemical stimuli, such as isopropyl‐β‐D‐thiogalactopyranoside (IPTG) gradients, demonstrates that the chemical environment has a substantial impact; filamentous cells aligned along the creases. These results illustrate the efficacy of parallel concentric wrinkled PDMS patterns in establishing regulated conditions for investigating microbial behavior and represent significant contributions to the fields of microbiology and biomedicine.

Reliable plasmonic biosensing with atmospheric pressure dielectric barrier discharge oxygen plasma treated polydimethylsiloxane microfluidic channels

Polydimethylsiloxane (PDMS) has emerged as a popular choice of material for many microfluidic and flexible sensing devices despite its inherent hydrophobic nature. Here, we report on the use of atmospheric pressure dielectric barrier discharge (APDBD) oxygen (O2) plasma in the development of hydrophilic and protein repulsive PDMS microchannels for plasmonic sensing application. The effect of plasma treatment on the wettability, surface energy and hydrophilicity retention capacity of PDMS was determined using contact angle measurements. The PDMS sample exposed to low-power DBD O2 plasma for 6 min demonstrated lowest contact angle of 35.15˚, surface energy of 62.83 mN/m and hydrophilicity retention capacity of more than 50 days in Phosphate Buffered Saline (PBS). The treatment of PDMS with plasma induces surface hydrophilicity due to the introduction of polar functional groups as evinced by Fourier Transform Infrared (FTIR) analysis without compromising the bulk structure and optical properties of PDMS which were confirmed by X-Ray Diffraction (XRD) and Ultraviolet-visible (UV-Vis) Spectroscopic measurements. The plasma treated PDMS was used in the construction of a microfluidic Surface Plasmon Resonance (SPR) biosensor which demonstrated very low detection limits of 7.13 ng/ml, 5.91 ng/ml and 1.47 ng/ml and high sensitivities of 16.8˚/(µg/ml), 13.5˚/(µg/ml) and 16.65˚/(µg/ml) against anti-Human Immunoglobulin-G protein raised in mouse, goat and rabbit respectively. The obtained results infer that APDBD O2 plasma is a promising technique to render PDMS hydrophilic suited for reliable microfluidic based plasmonic biosensing applications.

A New Silicon Mold Process for Polydimethylsiloxane Microchannels.

As an alternative to SU-8 soft lithography, a new silicon mold process of fabricating PDMS microchannel chips was proposed. A picosecond laser is used to cut through a 550 μm thick silicon wafer and generate the original microchannel pattern with a 50 μm minimum feature size. This single-crystal silicon pattern, with the edge debris caused by laser cutting being trimmed off by a KOH solution and with the protection field oxide layer being removed by BOE afterwards, firmly resided on a glass substrate through the anodic bonding technique. Four-inch wafers with microchannel patterns as the PDMS mold cores were successfully bonded on Pyrex 7740 or Eagle XG glass substrates for the follow-up PDMS molding/demolding process. This new maskless process does not need a photolithography facility, but the laser cutting service must be provided by professional off-campus companies. One PDMS microchannel chip for particle separation was shown as an example of what can be achieved when using this new process.

Lubricant-Coated Organ-on-a-Chip for Enhanced Precision in Preclinical Drug Testing.

In drug discovery, human organ-on-a-chip (organ chip) technology has emerged as an essential tool for preclinical testing, offering a realistic representation of human physiology, real-time monitoring, and disease modeling. Polydimethylsiloxane (PDMS) is commonly used in organ chip fabrication owing to its biocompatibility, flexibility, transparency, and ability to replicate features down to the nanoscale. However, the porous nature of PDMS leads to unintended absorption of small molecules, critically affecting the drug response analysis. Addressing this challenge, the precision drug testing organ chip (PreD chip) is introduced, an innovative platform engineered to minimize small molecule absorption while facilitating cell culture. This chip features a PDMS microchannel wall coated with a perfluoropolyether-based lubricant, providing slipperiness and antifouling properties. It also incorporates an ECM-coated semi-porous membrane that supports robust multicellular cultures. The PreD chip demonstrates its outstanding antifouling properties and resistance to various biological fluids, small molecule drugs, and plasma proteins. In simulating the human gut barrier, the PreD chip demonstrates highly enhanced sensitivity in tests for dexamethasone toxicity and is highly effective in assessing drug transport across the human blood-brain barrier. These findings emphasize the potential of the PreD chip in advancing organ chip-based drug testing methodologies.

Pre-enrichment-free detection of hepatocellular carcinoma-specific ctDNA via PDMS and MEMS-based microfluidic sensor

The growing interest in microfluidic biosensors has led to improvements in the analytical performance of various sensing mechanisms. Although various sensors can be integrated with microfluidics, electrochemical ones have been most commonly employed due to their ease of miniaturization, integration ability, and low cost, making them an established point-of-care diagnostic method. This concept can be easily adapted to the detection of biomarkers specific to certain cancer types. Pathological profiling of hepatocellular carcinoma (HCC) is heterogeneous and rather complex, and biopsy samples contain limited information regarding the tumor and do not reflect its heterogeneity. Circulating tumor DNAs (ctDNAs), which can contain information regarding cancer characteristics, have been studied tremendously since liquid biopsy emerged as a new diagnostic method. Recent improvements in the accuracy and sensitivity of ctDNA determination also paved the way for genotyping of somatic genomic alterations. In this study, three-electrode (Au-Pt–Ag) glass chips were fabricated and combined with polydimethylsiloxane (PDMS) microchannels to establish an electrochemical microfluidic sensor for detecting c.747G > T hotspot mutations in the TP53 gene of ctDNAs from HCC. The preparation and analysis times of the constructed sensor were as short as 2 h in total, and a relatively high flow rate of 30 µl/min was used during immobilization and hybridization steps. To the best of our knowledge, this is the first time a PDMS-based microfluidic electrochemical sensor has been developed to target HCC ctDNAs. The system exhibited a limit of detection (LOD) of 24.1 fM within the tested range of 2–200 fM. The sensor demonstrated high specificity in tests conducted with fully noncomplementary and one-base mismatched target sequences. The developed platform is promising for detecting HCC-specific ctDNA at very low concentrations without requiring pre-enrichment steps.Graphical

Effects of Tween 20 addition on electrokinetic transport in a polydimethylsiloxane microchannel.

Tween 20 is frequently added to particle suspensions for reducing the particle-wall adhesion and particle-particle aggregation in microfluidic devices. However, the influences of Tween 20 on the fluid and particle behaviors have been largely ignored. We present in this work the first experimental study of the effects of Tween 20 addition on the electrokinetic transport of fluids and particles in a polydimethylsiloxane microchannel. We find that adding 0.1% v/v Tween 20 to a buffer solution can significantly reduce the electroosmotic mobility as well as the electrokinetic and electrophoretic mobilities of polystyrene particles and yeast cells. Further increasing the Tween 20 concentration within the range typically used in microfluidic applications continues reducing these mobility values, but at a smaller rate. Our finding suggests that Tween 20 should be used with care in electrokinetic microdevices when the flow rate or particle/cell throughput is an important parameter.

Enhancing fabrication of hybrid microfluidic devices through silane‐based bonding: A focus on polydimethylsiloxane‐cyclic olefin copolymer and PDMS‐lithium niobate

AbstractEffective manipulation and control of fluids in microfluidic channels requires robust bonding between the different components. Polydimethylsiloxane (PDMS) is widely employed in microchannel fabrication due to its affordability, biocompatibility, and straightforward fabrication process. However, PDMS's low surface energy poses challenges in bonding with many organic and inorganic substrates, hindering the development of hybrid microfluidic devices. In this study, a simple and versatile three step process is presented for bonding PDMS microchannels with organic (cyclic olefin copolymer (COC)) and inorganic substrates (lithium niobate (LiNbO3)) using plasma activation and a silane coupling agent. Initially, the PDMS surface undergoes oxygen/argon plasma activation, followed by functionalization with (3‐aminopropyl) triethoxysilane (APTES). Subsequently, the COC or LiNbO3 is plasma activated and brought into contact with PDMS under a load at a specific temperature. Characterization by Fourier transform infrared, scanning electron microscopy, atomic force microscopy, and contact angle measurements confirmed the successful treatment of the substrates. In addition, bonding strength of the fabricated hybrid devices was assessed through leakage and tensile tests. Under optimized conditions (100°C and 4% v/v APTES), PDMS‐COC hybrid microchannels achieved a flow rate of 600 mL/h without leakage and a tensile strength of 562 kPa. Conversely, the PDMS‐ LiNbO3 assembly demonstrated a flow rate of 216 mL/h before leakage, with a tensile strength of 334 kPa. This bonding method exhibits significant potential and versatility for various materials in microfluidic applications, ranging from biomedical research to enhanced oil recovery.

On-chip light sheet illumination for nanoparticle tracking in microfluidic channels.

A simple and inexpensive method is presented to efficiently integrate light sheet illumination in a microfluidic chip for dark-field microscopic tracking and sizing of nanoparticles. The basic idea is to insert an optical fiber inside a polydimethylsiloxane (PDMS) elastomer microfluidic chip and use it as a cylindrical lens. The optical fiber is in this case no longer seen as only an optical waveguide but as a ready-made micro-optical component that is inexpensive and easy to source. Upon insertion, the optical fiber stretches the PDMS microchannel walls, which has two effects. The first effect is to tone down the intrinsic ripples in the PDMS that would otherwise create inhomogeneities in the light sheet illumination. The second effect is to remove any obliqueness of the channel wall and constrain it to be strictly perpendicular to the propagation of the illumination, avoiding the formation of a prismatic diopter. Through calculations, numerical simulations and measurements, we show that the optimal configuration consists in creating a slowly converging light sheet so that its axial thickness is almost uniform along the tracked area. The corresponding thickness was estimated at 12 μm, or 10 times the depth of field of the optical system. This leads to an at least six-fold increase in the signal-to-noise ratio compared to the case without the cylindrical lens. This original light-sheet configuration is used to track and size spherical gold nanoparticles with diameters of 80 nm and 50 nm.

Local Microbubble Removal in Polydimethylsiloxane Microchannel by Balancing Negative and Atmospheric Pressures.

Long-term experiments using organoids and tissues are crucial for drug development. Microfluidic devices have been regularly used in long-term experiments. However, microbubbles often form in these devices, and they may damage and starve cells. A method involving the application of negative pressure has been reported to remove microbubbles from microfluidic devices composed of polydimethylsiloxane; however, negative pressure affects the cells and tissues in microfluidic devices. In this study, a local microbubble removal method was developed using a microfluidic device with 0.5 mm thin polydimethylsiloxane sidewalls. The thin sidewalls counterbalanced the negative and atmospheric pressures, thereby localizing the negative pressure near the negatively pressurized chamber. Microbubbles were removed within 5 mm of the negatively pressurized chamber; however, those in an area 7 mm and more from the chamber were not removed. Using the local removal method, a long-term perfusion test was performed, and no contact was confirmed between the bubbles and the simulated tissue for 72 h.

Irreversible Bonding of Polydimethylsiloxane-Lithium Niobate using Oxygen Plasma Modification for Surface Acoustic Wave based Microfluidic Application: Theory and Experiment.

Acoustic microfluidic chips, fabricated by combining lithium niobate (LiNbO3) with polydimethylsiloxane (PDMS), practically find applications in biomedicine. However, high-strength direct bonding of LiNbO3 substrate with PDMS microchannel remains a challenge due to the large mismatching of thermal expansion coefficient at the interface and the lack of bonding theory. This paper elaborately reveals the bonding mechanisms of PDMS and LiNbO3, demonstrating an irreversible bonding method for PDMS-LiNbO3 heterostructures using oxygen plasma modification. An in-situ monitoring strategy by using resonant devices is proposed for oxygen plasma, including quartz crystal microbalance (QCM) covered with PDMS and surface acoustic wave (SAW) fabricated by LiNbO3. When oxygen plasma exposure occurs, surfaces are cleaned, oxygen ions are implanted, and hydroxyl groups (-OH) are formed. Upon interfaces bonding, the interface will form niobium-oxygen-silicon covalent bonds to realize an irreversible connection. A champion bonding strength is obtained of 1.1 MPa, and the PDMS-LiNbO3 acoustic microfluidic chip excels in leakage tests, withstanding pressures exceeding 60 psi, outperforming many previously reported devices. This work addresses the gap in PDMS-LiNbO3 bonding theory and advances its practical application in the acoustic microfluidic field.

Co-effect of hydrophobicity and cavities on flow characteristics at microscale

Flowing characteristics of drag reduction are experimentally and numerically investigated with a combination of hydrophobic coating and various cavities in polydimethylsiloxane microchannels. Two typical types of cavities with different geometrical parameters, rectangular and triangular, are studied with a wide range of Reynolds numbers from 0 to 300. A promising hydrophobic processing method is proposed based on a comprehensive performance on surface morphology, water repellency, and structural distortion. In this method, hydrofluoric acid (40 wt. %) is adopted and the corrosion time is set to 5 min. The velocity field and streamlines are acquired by the micro-particle image velocimetry system and numerical models to explain the flow patterns in particular. The slip length is measured as 13.38 μm in the hydrophobic straight channel. For rectangular cavities, the drag reduction rate reaches nearly 14.1% under no-slip condition and 33.2% under slip condition. A critical turning point of the co-effect is found by numerical results when the slip length is about 15 μm, which is also determined by the cavities. The convergent and divergent angles of triangular cavities play a critical role in the pressure drop due to the competition of the vortex and flow impingement. A nonlinear model is developed based on the numerical results to predict Poiseuille number with the relevant important variables for a two-dimensional microchannel. Our results reveal the fundamental physics of flowing characteristics with the co-design of hydrophobicity and microstructures, predicting a composite design method for widespread applications in microfluids.

Sensitivity of acoustofluidic particle manipulation to microchannel height in standing surface acoustic wave-based microfluidic devices

In this paper, the flow and particle trajectories, induced by standing surface acoustic waves (SSAWs) in a poly-dimethylsiloxane microchannel, are investigated by establishing a two-dimensional cross-sectional model with the finite element method and improved boundary conditions. Extensive parametric studies are conducted regarding the channel height, ranging from 0.2 to 4.0 times the spacing of the repetitive vertical interference pattern, to investigate its influences on the flow field and microparticle aggregation. The first-order flow field is found to be related to the channel height, exhibiting a periodic spatial distribution and oscillatory variation in its amplitude as the height changes. We theoretically analyze the propagation mechanism of the acoustic waves in the vertical direction and thus determine the periodicity of the wave interference pattern. Furthermore, we find that the speed of the particle aggregation is a function of the channel height, so the channel height can be optimized to maximize the strength of the first-order flow field and thus minimize the time of particle aggregation. The optimum heights can reduce the aggregation time by up to 76%. In addition, the acoustophoretic motions of microparticles exhibit a spatially dependent pattern when the channel height becomes larger than a quarter of the wavelength of the SAW, which can be explained by the change in the ratio between the radiation force and the streaming drag force from position to position. Our findings provide guidelines to the design and optimization of SSAW-based acoustofluidic devices.

Single-cell trapping and retrieval in open microfluidics

Among various single-cell analysis platforms, hydrodynamic cell trapping systems remain relevant because of their versatility. Among those, deterministic hydrodynamic cell-trapping systems have received significant interest; however, their applications are limited because trapped cells are kept within the closed microchannel, thus prohibiting access to external cell-picking devices. In this study, we develop a hydrodynamic cell-trapping system in an open microfluidics architecture to allow external access to trapped cells. A technique to render only the inside of a polydimethylsiloxane (PDMS) microchannel hydrophilic is developed, which allows the precise confinement of spontaneous capillary flow in the open-type microchannel with a width on the order of several tens of micrometers. Efficient trapping of single beads and single cells is achieved, in which trapped cells can be retrieved via automated robotic pipetting. The present system can facilitate the development of new single-cell analytical systems by bridging between microfluidic devices and macro-scale apparatus used in conventional biology.

The Effect of Temperature on the 3D printed Mold Using Polyethylene terephthalate glycol (PETG), Polylactic Acid (PLA) and Stereolithography (SLA)

The usage of 3D printing technology is explored in biomedical applications, specifically for drug delivery systems or cell separation using microfluidic channels. The fabrication of a 3D mold is very crucial, especially for fabricating a Polydimethylsiloxane (PDMS) microfluidic channel. This paper highlights the effect of temperature on the mold using different material elements, which is filament and resin, for fabricating a PDMS microfluidic channel. The mold structure was observed, and the mold was tested by repeating the process ten times. The molds were pre-heated, and the result shows that the structure of PLA mold gives the highest increases in sizing compared to PETG and SLA. The PDMS microchannel fabricated using 3D mold also shows the same result. The sizing of the PDMS microchannel increased linearly as it was tested five times with the same procedure. The mold was successfully fabricated with different materials, and the PDMS microchannel was successfully fabricated using the 3D-printed mold.

Irreversible PDMS bonding using flame activation of adhesives for fabrication of microfluidic and organ-on-chip devices

Polydimethylsiloxane (PDMS) is widely used for microfluidics fabrication in many disciplines due to its ease of use in soft lithography and its ability to bond liquid-tight seals. A variety of PDMS-PDMS bonding methods exist, but each may have limitations for some applications. For example, chemical bonding via oxygen plasma treatment is reliable but requires expensive equipment and specialized training. Here we present a rapid, low-cost, and accessible method for irreversible PDMS bonding in which flame treatment activates PDMS and Nitto 5302A adhesive surfaces. Using this technique, PDMS microchannels can be fabricated with a bonding integrity of up to 325 kPa burst pressure. This technique is suitable for fabricating organ-on-chip devices that support cell viability and establishment of key cellular features in cell line and primary cell cultures while maintaining bonding integrity.

Wax-shaped wettability assisted patterning of silver nanowires on various substrates as transparent, flexible, or stretchable electrodes

With the rapid development of flexible and wearable devices, flexible transparent conductive electrodes are highly demanded. However, there is no available technology that can pattern emerging electric materials (e.g., silver nanowires (AgNWs)) on versatile substrates in large scale to achieve high performance in different aspects on demands, such as transparency, flexibility, stretchability, and biocompatibility. Here, a wax-shaped wetting/dewetting (WSWD) method is demonstrated to pattern AgNWs networks into transparent conductive circuits ranging from tens of micrometers to centimeters on various substrates. For instance, the AgNWs coatings patterned on polyethylene terephthalate (PET) films achieve high electrical conductivity and optical transparency up to 73%, and a stable resistance (CV < 0.1%) after cyclic bending (>200 times); the AgNWs coatings patterned into polydimethylsiloxane (PDMS) microchannels exhibit good durability after cyclic bending (>200 times) and stretching (30% strain). Finally, the flexible AgNWs coatings are used as wearable transparent heaters applied to the human finger joint and wrist, which are thermally stable and safe (42 °C < T < 50 °C for 3 min) under cyclic bending. Thus, this method provides a simple, low-cost, green, and versatile means for fabricating transparent conductive electrodes, which can be applied to optoelectronic, wearable, and healthcare devices.

Fluoropolymer Functionalization of Organ-on-Chip Platform Increases Detection Sensitivity for Cannabinoids.

Microfluidic technology is applied across various research areas including organ-on-chip (OOC) systems. The main material used for microfluidics is polydimethylsiloxane (PDMS), a silicone elastomer material that is biocompatible, transparent, and easy to use for OOC systems with well-defined microstructures. However, PDMS-based OOC systems can absorb hydrophobic and small molecules, making it difficult and erroneous to make quantitative analytical assessments for such compounds. In this paper, we explore the use of a synthetic fluoropolymer, poly(4,5-difluoro-2,2-bis(trifluoromethyl)-1,3-dioxole-co-tetrafluoroethylene) (Teflon™ AF 2400), with excellent "non-stick" properties to functionalize OOC systems. Cannabinoids, including cannabidiol (CBD), are classes of hydrophobic compounds with a great potential for the treatment of anxiety, depression, pain, and cancer. By using CBD as a testing compound, we examined and systematically quantified CBD absorption into PDMS by means of an LC-MS/MS analysis. In comparison to the unmodified PDMS microchannels, an increase of approximately 30× in the CBD signal was detected with the fluoropolymer surface modification after 3 h of static incubation. Under perfusion conditions, we observed an increase of nearly 15× in the CBD signals from the surface-modified microchannels than from the unmodified microchannels. Furthermore, we also demonstrated that fluoropolymer-modified microchannels are compatible for culturing hCMEC/D3 endothelial cells and for CBD perfusion experiments.

Novel template digestive fabrication of multi-helical flow reactors

Embedded template-assisted fabrication by template removal technique is a simple and cost-effective method for preparation of complex 2D and 3D microchannels in soft polydimethylsiloxane (PDMS) platforms. However, employing this technique for fabrication of macroscale flow channels is impractical due to the requirement of a high swelling ratio of the PDMS network and other difficulties arising with the usage of millimeter-sized monofilament threads. The current work introduces a novel template digestion technique for fabricating topologically complex and transparent flow channels. The technique involves preparation of templates using flexible polyurethane (PU) tubes and their subsequent digestion by hot dimethyl sulfoxide (DMSO) within the embedding PDMS network. It is demonstrated for fabrication of various multi-helical channels (MHCs) with different geometrical features and compact modules of multiple MHCs which cater to process integration and intensification operations. Finally, an integrated operation of liquid-liquid extraction with subsequent phase separation is also demonstrated through typical parallel flow observed in MHCs.

Experimental and numerical investigations of heat transfer and flow characteristics during flow boiling in straight and diverging PDMS microchannel

Recent trends of miniaturization and densely packed circuits have necessitated the development of novel cooling methods, especially for emerging fields like flexible microelectronics, biotechnology, and nanotechnology. A polydimethylsiloxane (PDMS) based microchannel flow boiling heat sink can be used for these applications due to its unique properties as such as unique mechanical properties, biocompatibility, optical transparency, etc. PDMS microchannels are fabricated using soft lithography techniques, which involve using a mould to pattern the PDMS material. The mould is created using photolithography, and the PDMS is poured over the mould and cured to create the microchannels. In this study, an effort is made to compare the flow and heat transfer performance of a PDMS microchannel heat sink with different channel shapes, i.e. straight and diverging microchannel heat sinks for different heat flux and mass flux conditions. The PDMS microchannel was fabricated using the soft lithography technique and a flexible laser-induced graphene heater was fabricated and integrated with the developed PDMS microchannel to act as a heat source. Experimentally, the parameters like bubble pattern, pressure drop, exit vapour quality, and corresponding heat transfer coefficient were determined at varying mass and heat flux ranging from 19 to 114 kg m−2s−1 and from 13.3 to 156.6 kW m−2 respectively for both the microchannels. It was observed that the flow in the diverging microchannel is more stable and has a 13% higher heat transfer coefficient value compared to the straight microchannel under similar conditions. Further, a 3D numerical study was carried out to corroborate and elaborate on the results. The heat transfer, flow characteristics, and bubble pattern results show an excellent agreement with experimental results. This work has significant relevance in designing flexible microchannel heat sinks.