www.ietdl.org Published in IET Nanobiotechnology Received on 21st July 2012 Revised on 7th October 2012 Accepted on 9th October 2012 doi: 10.1049/iet-nbt.2012.0023
ISSN 1751-8741
Experimental investigation of magnetically actuated separation using tangential microfluidic channels and magnetic nanoparticles Ahsan Munir1, Zanzan Zhu1, Jianlong Wang 2, Hong Susan Zhou1 1
Department of Chemical Engineering, Worcester Polytechnic Institute, 100 Institute Road, Worcester, MA 01609, USA College of Food Science and Engineering, Northwest A&F University, 28 Xinong Road, Yangling, Shaanxi 712100, People’s Republic of China E-mail: [email protected]
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Abstract: A novel continuous switching/separation scheme of magnetic nanoparticles (MNPs) in a sub-microlitre fluid volume surrounded by neodymium permanent magnet is studied in this work using tangential microfluidic channels. Polydimethylsiloxane tangential microchannels are fabricated using a novel micromoulding technique that can be done without a clean room and at much lower cost and time. Negligible switching of MNPs is seen in the absence of magnetic field, whereas 90% of switching is observed in the presence of magnetic field. The flow rate of MNPs solution had dramatic impact on separation performance. An optimum value of the flow rate is found that resulted in providing effective MNP separation at much faster rate. Separation performance is also investigated for a mixture containing non-magnetic polystyrene particles and MNPs. It is found that MNPs preferentially moved from lower microchannel to upper microchannel resulting in efficient separation. The proof-of-concept experiments performed in this work demonstrates that microfluidic bioseparation can be efficiently achieved using functionalised MNPs, together with tangential microchannels, appropriate magnetic field strength and optimum flow rates. This work verifies that a simple low-cost magnetic switching scheme can be potentially of great utility for the separation and detection of biomolecules in microfluidic lab-on-a-chip systems.
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Introduction
Biomolecular separation using microfluidics technology involves capture, isolation and release of target biomolecules from impure samples. It is used for purification, pre-concentration and detection of biomolecules [1, 2], proteins [3, 4], DNA [5, 6] and cells [7, 8] in clinical diagnostics, drug discovery and microbiology. There are a number of techniques currently available on microfluidic platforms for particle separation, such as electrophoresis [9, 10], dielectrophoresis [11], size-based separation [12–14], pinched flow fractionation [15, 16], acoustic separation [17], inertial separation [18, 19] and magnetic bioseparation [8, 20–25]. Among these techniques, magnetically actuated methods seem to be more promising because of the simplicity of design and ease of operation. This method utilises surface-functionalised magnetic particles to trap target biomolecules through specific chemical binding followed by separation using magnetic manipulation. Magnetic bioseparation technique is dependent on the interaction of chemical bonds and therefore allows highly specific and selective biomolecular separation when compared with other techniques that rely on geometrical or physical properties of the species.
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Most of the magnetic bioseparation systems developed on microfluidic platform are based on magnetic micro-sized particles [2, 8, 26–28], however, less intensively studied microfluidic bioseparation [29–33] schemes are the emerging nanoscale magnetic particles. Compared with microparticles, nanoscale materials possess better properties that can be advantageously deployed in microfluidic devices. Higher surface to volume ratio [34] makes magnetic nanoparticles (MNPs) nearly ideal for the manipulation and detection of attached biomolecules [30, 35]. For example, it has been shown that functionalised MNPs have enhanced the detection of small molecules using surface plasmon resonance (SPR) spectroscopy [36]. The small size of MNPs causes minimal disturbance to the attached biomolecules [34] as well as provides enhanced interaction for chemical binding and tagging. Most importantly, they are super-paramagnetic in nature [34], that is, their magnetisation without a magnetic field is zero. The super-paramagnetic nature ensures that they stay suspended in carrier liquid when the magnetic field is removed. Unlike micrometre-sized particles, these particles do not irreversibly agglomerate or precipitate and can be repeatedly used subjected to magnetic fields of varying strength without causing any adverse effects. This also makes it easy for the removal or capture of tagged biomolecules of
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www.ietdl.org interest once the magnetic field is removed. Overall, the inclusion of MNPs in microfluidic devices for biomolecule separation, manipulation and detection will not only enhance the device functionality and separation performance but also broaden the utility of these devices in real-world applications. Separation of biomolecules using magnetic field actuation can be achieved either in conventional batch process or in continuous flow mode [37–40]. In the batch process [24, 41, 42], the magnetic particles tagged with target biomolecules are trapped or retained using magnetic field and subsequently released, after the removal of non-targets. A number of devices have been developed with various magnet designs [1, 28, 43] to accomplish magnetic bioseparation. Useful batch mode operation suffers from low separation efficiency, longer incubation and handling time, and results in significant contamination due to non-specific binding of impurities with magnetic beads [44]. More importantly, their incorporation in point-of-care microfluidic testing devices will require more complicated multi-step fluidic handling. On the other hand, continuous flow magnetic bioseparation processes overcome the above limitations because they employ magnetic fractionation, that is, continuous accumulative deflection of magnetic particles tagged to biomolecules. This method does not require multistep fluidic handling; moreover, higher magnetic field is also not a requisite because the process only depends on deflection rather than on complete trapping of MNPs. Continuous flow magnetic bioseparation can be distinguished into two types: one in which electromagnets or magnetic microstrips typically of alloy or ferromagnetic materials are integrated on the device substrate to generate a magnetic field gradient that deflects magnetic beads [22, 25, 45–47]. Substantial cost and effort is required to design and fabricate these systems. Alternatively, in the second type, a simple external permanent magnet assembly is used that provides greater flexibility and simplicity in device design to achieve higher magnetic field-assisted bioseparation [48]. The development of continuous flow magnetic bioseparation scheme together with a simple low-cost approach for selective injection or removal of biomolecules bound to MNPs is highly desirable to complement the existing microfluidic technology available for bioseparation. In this work, a simple, low-cost and generic microfluidic platform is assembled to demonstrate continuous flow magnetic bioseparation using tangential microfluidic channels and MNPs. A major innovation of this set-up lies in the fabrication of tangential microchannel that acts as magnetic microfluidic switch to manipulate flows containing MNPs and can accomplish efficient bimolecular separation. Standard moulding process combined with a novel rapid prototyping method is used in this work to develop low-cost polydimethylsiloxane (PDMS) microchannel. The fabrication method used in this work circumvents the requirement for a clean room [49]. It also eliminates the combination of two pieces of element, such as in standard fabrication method where negative or positive stamp on PDMS is combined with glass or silicon using plasma. This further overcomes the issue of leakage in microfluidic channels. Continuous switching/separation of MNP in a sub-microlitre fluid volume surrounded by neodymium permanent magnet are studied. On the basis of MNPs concentration measurement using optical technique, separation efficiency is analysed for scenarios with and without magnetic field. Separation performance of the set-up is also studied for a mixture containing non-magnetic IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 102–110 doi: 10.1049/iet-nbt.2012.0023
polystyrene (PS) particles and MNPs. The effect of flow rate on continuous flow separation of MNPs in tangential microchannel is also investigated. This work demonstrates that a simple low-cost magnetic switching scheme using tangential microchannel together with MNPs can be potentially of great utility, which can further improve the functional performance of magneto-fluidic bioseparation systems.
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Experimental materials and methods Microchannel fabrication
Low-cost rapid micromoulding technique was used to develop 75 mm long and 800 µm diameter tangential microfluidic channels. The steps used in fabricating the microchannels are shown in Fig. 1. The first step in preparing these channels involved the development of mould, which was prepared using aluminium wires each of 800 µm diameters. The wires were placed at the centre and approximately at half the depth of the empty Petri dish such that they overlap each other at an angle of 30°. For that, small holes were drilled using the stainless steel syringe, which was later closed using adhesive tapes. It was also made sure that they touch each other approximately at the centre. PDMS (Sylgard 184, Dow Corning, USA) with a base and curing agent mixed in a ratio of 10:1 was poured onto the mould and was degassed to remove any bubbles using desiccators. The uncured PDMS was baked in an oven (65°C) for 1 h. The final step was to peel off the cured-PDMS containing the aluminium wires from the Petri dish. The sides of the cured PDMS were cut using a razor blade, leaving a significant amount of the wire exposed outside. With the help of pliers the wires were carefully removed. To make this process easier, the cured PDMS block containing the embedded wires was washed with acetone, which swelled the PDMS and expanded the channels prior to pulling out the wires. An internal access area was created at the centre of overlap where the wires touched each other making the only connection between two microchannels. The microchannels were then connected with the tygon tubing using the stainless tip obtained from microsyringe. The tip was inserted into the microchannels to make leakage-free connection. 2.2
Microfluidic system set-up
A simple experimental set-up to carry out magnetohydrodynamic experiments is shown in Fig. 2. As shown in the figure, the microfluidic channels are connected with inlet and outlet via flexible tygon tubing. Lower microchannel inlet (LT1) is used to transport MNP solution, whereas deionised (DI) water flows from the upper channel inlet (UT1). To provide leak-free connections, a microsyringe tips made of stainless steel are embedded into the microchannel inlet/outlet for secure connections between the flexible tubes and the microfluidic chip. A differential pressure drop is maintained inside the channels by connecting the outlet of the microchannels (LT2 and UT2) to peristaltic micropump (P625 Peristaltic Pump, Instech, USA) using an in-house developed PDMS T-shaped connector. The flow rates were varied using a precise bi-directional speed controller on the pump. This simple method allows for a good control of the flow in the channels in suction mode. In order to have more precise control over the flow, individual syringe pumps can be used 103
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Fig. 1 Fabrication step used in developing leak-proof microfluidic channels
Fig. 2 Experimental set-up of magnetic field based separation using tangential microchannels and MNPs Inset shows the close-up of tangential microchannel with neodymium magnet
for each microchannel instead of peristaltic micropump. The pumps can be calibrated in order to take into account a small imbalance in the flow resistances of the two channels. An upward magnetic pull force is obtained using the permanent neodymium magnets (Grade N52, KJ Magnetics, USA), as shown in Fig. 2 (inset), near the access hole where the upper microchannel is connected to the lower microchannel. The permanent magnet was 1 × 1 inch in dimensions with 3/8-inch thickness. The ‘residual flux density’ of this magnet was 14 800 Gauss with magnetisation along the thickness of the magnet. Optical images of the bioseparation experiments at different time points were obtained using the digital camera (Sony Cyber Shot DSC-W530, Sony Electronics Inc., USA). MNPs of 200 nm diameter (fluidMAG-ARA Chemicell GMBH, Germany) with a concentration of 1 mg/ml were transported through lower inlet. The MNPs consisted of an inner core made up of magnetite (Fe3O4) crystals of approximately 12 nm diameter, which were embedded in a biocompatible 104 & The Institution of Engineering and Technology 2014
polysaccharide matrix for better stability that also prevented biodegradation. The overall diameter of the nanoparticles was approximately 200 nm, whereas the volume fraction of magnetite within a composite particle is 80%. For different flow rates, the effluent was collected at the outlets once all the solution has passed through the microchannels. The volume collected at the outlet was regularly verified to confirm the equal flow rates in both the microchannels. The concentration of MNP solution in the effluent was estimated from in-house determined calibration curve. The calibration curves were generated from original stock of MNPs solution diluted at different concentrations. A dynamic slight scattering instrument Zetasizer Nano S (Malvern Zetasizer Nano S, UK) was used. The Zetasizer Nano S measures the intensity of scattered light of various concentrations of sample at one angle; this is compared with the scattering produced from a standard one (i.e. Toluene). In general, Zetasizer is used to measure the size of molecules based on the count rate, which is a fairly IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 102–110 doi: 10.1049/iet-nbt.2012.0023
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Fig. 3 Calibration curve obtained for MNPs using scattering intensity obtained from Zetasizer Nano S Inset shows different concentrations of MNPs used in generating calibration curve
stable value of the sample calculated from dynamic scattering. This value is used as a method of determining the relative concentration of a sample of stable size – as the count rate goes down, so does the concentration. While the Zetasizer software does not automatically estimates sample concentration, the count rate is used in this work to calculate concentration of samples. Power-law calibration curve of scattering intensity (kilocounts per second, kcps) against concentration of MNPs (mg/ml) were obtained for 200 nm particles (R 2 = 0.9908) (see Fig. 3). To obtain the concentration of MNPs coming out of the system under various condition of flow rate, the outlet samples from the effluent was taken in a cuvette and placed in the Zetasizer to obtain the unknown scattering intensity (kcps) of the sample. The calibration curve was used to convert the scattering intensity into concentration (mg/ml). Since the inlet concentration of MNPs was known, percentage separated was calculated.
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whereas at higher aspect ratio the fluid exchange is minimal and the fluid largely continues through the intersection within the same fluid stream. To minimise the exchange of fluid between microchannels, we tested many microchannels and found that microchannel with a diameter of 800 µm produced best results. The Reynolds number for the flow rate (0.6–11 µl/s) used in the experiments ranges from 0.239 to 4.42. For such a low Reynolds number, the inertial term in the Navier–Stokes equation can be neglected. This type of flow is referred as Stokes flow or Creeping flow and occurs in systems with small flow length scales. Based on the Reynolds number computation and experimental observation, the flow profile was always creeping instead of parabolic, and thus, the flow within the microchannels was always remained laminar. To investigate the transport of MNP solution in microchannels, the movement of the nanoparticles between the two tangential streams in the absence of any applied magnetic force is examined. The flow rates of the two channels were the same (∼5 µl/s). A 100 µl of MNP solution having a concentration of 1 mg/ml were injected in the lower microchannel, whereas DI-water flow was maintained in the upper microchannel. The transport of MNPs was recorded after every 2 s. It can be seen from Fig. 4 that after 6 s almost all the solution continues through the intersection within the same flow stream with a very small amount of solution transferring to upper stream because of diffusion across the interface.
Results and discussion
3.1 Qualitative and quantitative analysis of MNPs separation Magnetic manipulation and switching of nanoparticles between two flow streams is a complementary way of separating biomolecules or cells in microfluidic devices when these biomolecules are tagged with nanoparticles. It is based on the attraction of the nanoparticles tagged biomolecules to regions with higher magnetic field intensity. In this section, qualitative analysis of the switching or separation of MNPs is performed with the aid of imaging using a digital camera. The set-up of the experiment is already described in Section 2. In order to examine the MNP movement within the magnetic fields, tangential PDMS microchannel was fabricated using a simple and inexpensive benchtop fabrication method, as described earlier. It has been shown by Ismagilov et al. [50, 51] that the flow fields in tangential microchannels are independent of the contact area but strongly depends on the channel aspect ratio. A too low aspect ratio results in divergence of fluid from one channel into another channel, IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 102–110 doi: 10.1049/iet-nbt.2012.0023
Fig. 4 Flow of MNPs in the tangential microchannel in the absence of magnetic field, where 100 µl of MNP solution having a concentration of 1 mg/ml was injected in the lower microchannel 105
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www.ietdl.org In the next experiments, the movement of MNPs was investigated in the presence of magnetic field force. A neodymium magnet (N52) was aligned on top of the intersection of the two microchannels such that the edge of the magnet is very close to the intersection in order to provide maximum magnetic field force. The assembly of magnet is shown in Fig. 1 and described in Section 1.2 in more detail. It was found that the distance between the intersection of the two microchannels and the edge of the neodymium magnet was approximately 2 mm. The magnetic field intensity was computed using our in-house
developed COMSOL™ numerical code [21, 52], which were also validated using the well-developed analytical expressions given by Furlani et al. [38]. The magnetic flux density at this distance was computed to be in the range of 0.6–0.8 T. Mass transfer of 100 µl of MNP solution having a concentration of 1 mg/ml entering the lower microchannel was recorded after every 2 s. It can be seen from Fig. 5a that after 6 s MNPs preferentially move upwards because of the magnetic force acting on them near the intersection and flows with the upward stream. A significant amount of MNPs switching was achieved near the intersection that can
Fig. 5 MNPs switching between microfluidic channels using Neodymium magnet a Snapshot of tangential microchannel at different times. MNPs were injected in the lower microchannel b Close-up of tangential microchannel after 18 s shows MNPs switching 106 & The Institution of Engineering and Technology 2014
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www.ietdl.org be seen in Fig. 5b. A very small quantity of MNPs still emerges from lower stream, which could be due to moderate disturbance in flow field arising from imperfect channel geometry which causes roll up of some particles. In order to further investigate the performance of switching of MNPs, Zetasizer experiments were performed to evaluate the concentration of MNPs exiting both the outlets. The sample from the outlets is collected until all the solution has passed through the microchannel. It is taken in a cuvette and placed in the Zetasizer to obtain unknown scattering intensity (kcps) of the sample. Previously determined calibration curve is used to convert the scattering intensity into unknown outlet concentration (mg/ml). Each experiment both in the presence and absence of permanent magnet was performed in triplicates and the average values together with the standard deviation were reported. A 100 µl of MNP solution having a concentration of 1 mg/ml was injected into the lower microchannel with a flow rate of 5 µl/s. It can be seen from Fig. 5 that when the magnetic field is not deployed, most of the MNP follows the same flow stream with the concentration of MNPs exiting the lower microchanel was approximately 0.88 mg/ml. The concentration of MNPs found in the upper microchannel was very small ( ∼ 0.064 mg/ml approximately). This proves that no switching or separation takes place in the absence of magnetic field. It was also found that around 5.5% MNPs were not found either in the upper or lower microchannel. This could be due to the fact that some of the MNPs got trapped within the microchannel and never exited the system. Some of this error could also arise from instrumental error because of the correlation made between scattering intensity and concentration. However, when the magnetic field was used, as seen from data given in Fig. 6, 0.90 mg/ml of the MNPs were switched from lower microchannel to upper microchannel. This was because these MNPs experience magnetic pull force near the area of intersection and were transferred into upward flow stream. A very small amount ( ∼ 0.067 mg/ml) was found in the lower microchannel with approximately 3% not found either in the upper or lower microchannel. It was seen that out of 3% some of these MNPs got trapped on the inner walls of microchannel because of strong magnetic force. From the above analysis,
Fig. 6 Concentration of MNPs eluted from upper and lower microchannel when 100 µl of 1 mg/ml MNPs were injected through lower microchannel at flow rate of 5 µl/s in the absence and presence of magnetic field (Neodymium magnet, N52) IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 102–110 doi: 10.1049/iet-nbt.2012.0023
it can be demonstrated that 90% separation of MNPs was achieved using tangential microfluidic channels. 3.2
Effect of flow rate on MNPs separation
In this section, the effect of flow rate on the MNPs separation in tangential microchannel is investigated. Variation in solvent flow rates impacts the residence time of MNPs within the microchannels as well as the drag and magnetic forces acting on these particles. Although the magnetic force is independent of the flow rate, but the time that the magnetic force acts on the particles is related to the average residence time of the particles in the vicinity of the magnet. A longer residence time means smaller flow rates that translate to smaller drag forces; therefore if the particles are exposed to magnetic force for longer time, there will be a strong tendency that these particles will experience higher magnetic force than drag force and as a consequence will be pulled across the interface and into the other microchannel. To test the role of flow velocity on magnetic switching or separation, a series of experiments were performed using a constant magnetic field and varied flow rates. MNPs with an initial concentration of 1 mg/ml and volume of 100 µl were injected from the lower microchannel at varying flow rates. The concentration of MNPs that exited the intersection in the upper and lower microchannels was detected using Zetasizer instrument together with the procedure described in Section 1.1. Each experiments both in the presence and absence of permanent magnet was performed in triplicates and the average values together with the standard deviation were reported. Since the inlet concentration of MNPs was known, percentages of MNPs separated between two tangential microchannels were calculated. It can be seen from Fig. 7a, that switching of MNPs takes place when magnetic field is used. A higher percentage of MNPs were pulled from the lower microchannel to upper microchannel at lower flow velocity ( < 5 µl/s). However, when the flow velocity was increased beyond 5 µl/s, a linear decrease in MNP switching was observed. This is because at lower flow velocity, the drag force acting on the MNPs was small and the magnetic force acted for greater amount of time on the MNPs near the junction. To investigate quantitatively, the average residence time of MNPs at the junction region ( ∼ 800 µm) was calculated for different flow rates. It can be seen from Fig. 7b that at lower flow rates, the magnetic force acts for greater amount of time on MNPs ( ∼ 3000 ms) when they are passing the junction as compared with higher flow rates. This result in more number of MNPs pulled in the upper flow stream from lower microchannel, which was not the case when the flow rate was increased. Another interesting observation was made in the absence of magnetic field when the flow rates were smaller than 5 µl/s. It was found that at a very low flow rates ( ∼ 0.6 µl/s), small amount of MNPs ( ∼ 18%) were pulled in the upper channel. In order to test whether this behaviour was due to diffusional transport, an estimate for the diffusion coefficient was obtained using the Stokes–Einstein equation for the diffusivity given in (1). DMNP =
kB T 6phRMNP
(1)
where RMNP is the radius of the MNP that is diffusing, η is the viscosity of the suspending fluid (water), kB is the Boltzmann 107
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Fig. 7 Effect of flow rate on MNPs separation a Percentage of MNPs eluting from lower (O) and upper (Δ) microchannel at different flow rates in the presence and absence of magnetic field. MNPs initial concentration was 1 mg/ml and 100 µl of the sample was injected from lower microchannel b Residence time of MNPs in the junction region against flow rates. Error bar represent the standard deviation obtained after three injections
constant and T is the temperature. This formula is applicable for a spherical particle much larger than the solvent molecules. In this work, we assume MNPs as spherical particles suspended in water. Based on the above equation, the diffusion coefficient for 200 nm MNPs was calculated to be 2.14 × 10 − 12 m2/s. In order to further investigate, the Peclet number, Pe, which is the product of system length and fluid velocity, divided by diffusivity was computed. It was found that for all flow conditions, Pe was much greater than one implying that diffusion is not dominant and small migration of MNPs could be because of convective flow field that diverted near the imperfect junction boundaries. This trend was minimised as the flow rate was increased. A more thorough investigation will be needed to account for such observed behaviour, which is beyond the scope of this work. From these experiments it was illustrated that the application of magnetic field causes the MNPs to move from the lower microchannel to upper microchannel. However, the percentage of MNPs that can be magnetically as well as non-magnetically transported into the upper microchannel is dramatically influenced by the flow rate of both fluid streams. As the flow rate decreases from 11 to 0.6 µl/s under magnetic field, the percentage of MNPs eluting from the upper microchannel increases from 48.8 to 93.4% with more drastic change found in between flow rates of 5–11 µl/s. The improved separation achieved at lower flow rates was not simply a result of the increased residence time but is a function of both drag and magnetic force acting on these MNPs. By carefully calibrating the fluid flow, an optimum value of flow rate can be achieved to provide maximum switching of MNPs together with higher throughput essential for bioseparation application. The sizes of MNPs also play an important role in the separation process. In this work, only one type of MNPs was used. The magnetic force acting on MNPs is directly 108 & The Institution of Engineering and Technology 2014
proportional to size of MNPs; a larger diameter MNPs will result in higher magnetic forces, therefore the flow rate and strength of magnets can be adjusted to avoid capturing of particles on MNPs or even clogging microchannels. A more detailed optimisation study taking into account MNPs size, flow velocity, junction length and magnetic field strength is the next step before realising the advantage of using this concept. 3.3 Magnetic separation of a mixture of magnetic and non-magnetic particles Magnetically actuated switching of biomolecules tagged with MNPs from one fluid stream into another, while leaving behind non-magnetic particles, is an excellent strategy to achieve microfluidic-based separation of biomolecules continuously. This strategy was demonstrated and tested in this section. A mixture of 1 mg/ml of PS (60 nm) and 1 mg/ ml of MNPs (200 nm) with a 1:1 volume ratio was injected from the lower microchannel in the presence of magnetic field. The total volume of the mixture was 100 µl and the injection rate of 5 µl/s was maintained in both the upper and lower microchannels. Samples from the outlets were collected and analysed using Zetasizer. Both the size as well as concentration measurement was performed using the instrument. Figs. 8a and b give the histogram of the size distribution of the particles eluting from the upper and lower channels. It can be seen from the results that the average size of particles eluting from the upper and lower channels were around 273.1 and 68.75 nm, respectively. This illustrate that most of the MNPs were pulled into the upper microchannel because of magnetic force leaving behind non-magnetic PS particles in the lower microchannel. A slight error in size estimation could be due to presence of MNPs in lower and PS in upper IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 102–110 doi: 10.1049/iet-nbt.2012.0023
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Fig. 8 5 µl/s
Magnetic field-based separation of a mixture of PS (60 nm) and MNPs (200 nm) injected from lower microchannel at a flow rate of
a Average diameter of sample eluted from the upper microchannel b Average diameter of sample eluted from the lower microchannel c Percentage of MNPs eluted from the upper and lower microchannels d Percentage of PS eluted from the upper and lower microchannels
microchannel. Based on the particle size histograms, it is evident that the application of the magnetic field preferentially removed the MNPs from the lower microchannel. In order to investigate the separation or switching efficiency, the concentrations of MNPs and PS were computed using the method described in earlier sections. It can be seen from Figs. 8c and d that 89.9% of MNPs were pulled into the upper microchannel, whereas 90.6% of PS stayed in lower microchannel. From the above analysis, it is evident that magnetic field-assisted preferential switching of MNPs can be efficiently utilised in separating biomolecules in microfluidic devices.
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Conclusion
Magnetic fields can be effectively used to manipulate the movement of MNPs together with biomolecules attached on their surfaces. In this work a continuous switching/ separation of MNPs in a sub-microlitre fluid volume surrounded by neodymium permanent magnet is studied. A simple, low-cost and generic microfluidic platform is developed for proof-of-concept experimentation. On the basis of MNPs concentration measurement, the movement of the nanoparticles between the two tangential streams in the absence and presence of the applied magnetic force was investigated. It was found that negligible switching of MNPs takes place in the absence of magnetic field, whereas 90% of switching was observed when the magnetic field was employed. The flow rate of MNP solution had a dramatic impact on separation performance. A too high flow rate resulted in decrease in switching of MNPs, IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 102–110 doi: 10.1049/iet-nbt.2012.0023
whereas a too low flow rate did not significantly improve the separation efficiency. It was observed that by carefully calibrating the fluid flow, an optimum value of flow rate can be found to provide maximum switching of MNPs together with higher throughput essential for the bioseparation application. Separation performance was also studied for a mixture containing non-magnetic PS particles and MNPs. It was found that MNPs preferentially moved from lower microchannel to upper microchannel, resulting in an efficient separation from non-magnetic particles. The proof-of-concept experiments performed in this work further demonstrates that microfluidic-based separation of biomolecules can be efficiently achieved using functionalised MNPs, together with tangential microchannels, appropriate magnetic field strength and optimum flow rates. This work further demonstrates that a simple low-cost magnetic switching scheme can be potentially of great utility for the separation and detection of biomolecules and cells in lab-on-a-chip systems.
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Acknowledgments
The authors gratefully acknowledge the support from the National Science Foundation (grant no. CMMI-1030289) and National Natural Science Foundation of China (grant no. 31101274)
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References
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www.ietdl.org 2 Bu, M.Q., Christensen, T.B., Smistrup, K., Wolff, A., Hansen, M.F.: ‘Characterization of a microfluidic magnetic bead separator for high-throughput applications’, Sensors and Actuators A-Physical, 2008, 145, pp. 430–436 3 Hahn, Y.K., Jin, Z., Kang, J.H., et al.: ‘Magnetophoretic immunoassay of allergen-specific IgE in an enhanced magnetic field gradient’, Anal. Chem., 2007, 79, pp. 2214–2220 4 Hayes, M.A., Polson, N.A., Phayre, A.N., Garcia, A.A.: ‘Flow-based microimmunoassay’, Anal. Chem., 2001, 73, pp. 5896–5902 5 Lehmann, U., Vandevyver, C., Parashar, V.K., Gijs, M.A.M.: ‘Droplet-based DNA purification in a magnetic lab-on-a-chip’, Angew. Chem. – Int. Edn., 2006, 45, pp. 3062–3067 6 Yeung, S.W., Hsing, I.M.: ‘Manipulation and extraction of genomic DNA from cell lysate by functionalized magnetic particles for lab on a chip applications’, Biosens. Bioelectron., 2006, 21, pp. 989–997 7 Zheng, T., Yu, H.M., Alexander, C.M., Beebe, D.J., Smith, L.M.: ‘Lectin-modified microchannels for mammalian cell capture and purification’, Biomed. Microdevices, 2007, 9, pp. 611–617 8 Xia, N., Hunt, T.P., Mayers, B.T., et al.: ‘Combined microfluidic-micromagnetic separation of living cells in continuous flow’, Biomed. Microdevices, 2006, 8, pp. 299–308 9 Liu, F.K., Lin, Y.Y., Wu, C.H.: ‘Highly efficient approach for characterizing nanometer-sized gold particles by capillary electrophoresis’, Anal. Chim. Acta, 2005, 528, pp. 249–254 10 Xu, X.Y., Caswell, K.K., Tucker, E., Kabisatpathy, S., Brodhacker, K. L., Scrivens, W.A.: ‘Size and shape separation of gold nanoparticles with preparative gel electrophoresis’, J. Chromatograph. A, 2007, 1167, pp. 35–41 11 Durr, M., Kentsch, J., Muller, T., Schnelle, T., Stelzle, M.: ‘Microdevices for manipulation and accumulation of micro and nanoparticles by dielectrophoresis’, Electrophoresis, 2003, 24, pp. 722–731 12 Yamada, M., Kano, K., Tsuda, Y., et al.: ‘Microfluidic devices for size-dependent separation of liver cells’, Biomed. Microdevices, 2007, 9, pp. 637–645 13 Chen, X., Cui, D.F., Liu, C.C., Li, H., Chen, J.: ‘Continuous flow microfluidic device for cell separation, cell lysis and DNA purification’, Anal. Chim. Acta, 2007, 584, pp. 237–243 14 Pamme, N., Wilhelm, C.: ‘Continuous sorting of magnetic cells via on-chip free-flow magnetophoresis’, Lab Chip, 2006, 6, pp. 974–980 15 Jain, A., Posner, J.D.: ‘Particle dispersion and separation resolution of pinched flow fractionation’, Anal. Chem., 2008, 80, pp. 1641–1648 16 Yamada, M., Nakashima, M., Seki, M.: ‘Pinched flow fractionation: continuous size separation of particles utilizing a laminar flow profile in a pinched microchannel’, Anal. Chem., 2004, 76, pp. 5465–5471 17 Nam, J., Lim, H., Kim, D., Shin, S.: ‘Separation of platelets from whole blood using standing surface acoustic waves in a microchannel’, Lab Chip, 2011, 11, pp. 3361–3364 18 Di Carlo, D., Irimia, D., Tompkins, R.G., Toner, M.: ‘Continuous inertial focusing, ordering, and separation of particles in microchannels’. Proc. Natl. Acad. Sci. USA, 2007, 104, pp. 18892–18897 19 Kuntaegowdanahalli, S.S., Bhagat, A.A.S., Kumar, G., Papautsky, I.: ‘Inertial microfluidics for continuous particle separation in spiral microchannels’, Lab Chip, 2009, 9, pp. 2973–2980 20 Choi, J.W.: ‘Fabrication of micromachined magnetic particle separators for bioseparation in microfluidic systems’, in: ‘Methods in molecular biology’ (H.P. Inc., Totowa, NJ, 2006, 2nd edn.), vol. 321, pp. 65–81 21 Munir, A., Wang, J., Zhou, H.S.: ‘Dynamics of capturing process of multiple magnetic nanoparticles in a flow through microfluidic bioseparation system’, IET Nanobiotechnol., 2009, 3, pp. 55–64 22 Adams, J.D., Kim, U., Soh, H.T.: ‘Multitarget magnetic activated cell sorter’. Proc. Natl. Acad. Sci. USA, 2008, 105, pp. 18165–18170 23 Bucak, S., Jones, D.A., Laibinis, P.E., Hatton, T.A.: ‘Protein separations using colloidal magnetic nanoparticles’, Biotechnol. Prog., 2003, 19, pp. 477–484 24 Earhart, C.M., Wilson, R.J., White, R.L., Pourmand, N., Wang, S.X.: ‘Microfabricated magnetic sifter for high-throughput and high-gradient magnetic separation’, J. Magn. Magn. Mater., 2009, 321, pp. 1436–1439 25 Inglis, D.W., Riehn, R., Austin, R.H., Sturm, J.C.: ‘Continuous microfluidic immunomagnetic cell separation’, Appl. Phys. Lett., 2004, 85, pp. 5093–5095 26 Pamme, N.: ‘Magnetism and microfluidics’, Lab Chip, 2006, 6, pp. 24–38 27 Smistrup, K., Lund-Olesen, T., Hansen, M.F., Tang, P.T.: ‘Microfluidic magnetic separator using an array of soft magnetic elements’, J. Appl. Phys., 2006, 99, (8), pp. 08P102–08P102-3
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28 Ahn, C.H., Allen, M.G., Trimmer, W., Jun, Y.N., Erramilli, S.: ‘A fully integrated micromachined magnetic particle separator’, J. Microelectromech. Syst., 1996, 5, pp. 151–158 29 Shih, P.H., Shiu, J.Y., Lin, P.C., Lin, C.C., Veres, T., Chen, P.: ‘On chip sorting of bacterial cells using sugar-encapsulated magnetic nanoparticles’, J. Appl. Phys., 2008, 103, pp. 07A316–07A316-3 30 Pankhurst, Q.A., Connolly, J., Jones, S.K., Dobson, J.: ‘Applications of magnetic nanoparticles in biomedicine’, J. Phys. D, Appl. Phys., 2003, 36, pp. R167–R181 31 Kell, A.J., Somaskandan, K., Stewart, G., Bergeron, M.G., Simard, B.: ‘Superparamagnetic nanoparticle-polystyrene bead conjugates as pathogen capture mimics: a parametric study of factors affecting capture efficiency and specificity’, Langmuir, 2008, 24, pp. 3493–3502 32 Schneider, T., Karl, S., Moore, L.R., Chalmers, J.J., Williams, P.S., Zborowski, M.: ‘Sequential CD34 cell fractionation by magnetophoresis in a magnetic dipole flow sorter’, Analyst, 2010, 135, pp. 62–70 33 Williams, P.S., Carpino, F., Zborowski, M.: ‘Characterization of magnetic nanoparticles using programmed quadrupole magnetic field-flow fractionation’, Philos. Trans. R. Soc. Math. Phys. Engng. Sci., 2010, 368, pp. 4419–4437 34 Gijs, M.A.M.: ‘Magnetic bead handling on-chip: new opportunities for analytical applications’, Microfluidics Nanofluidics, 2004, 1, pp. 22–40 35 Berry, C.C., Curtis, A.S.G.: ‘Functionalisation of magnetic nanoparticles for applications in biomedicine’, J. Phys. D Appl. Phys., 2003, 36, pp. R198–R206 36 Wang, J.L., Munir, A., Zhu, Z.Z., Zhou, H.S.: ‘Magnetic nanoparticle enhanced surface plasmon resonance sensing and its application for the ultrasensitive detection of magnetic nanoparticle-enriched small molecules’, Anal. Chem., 2010, 82, pp. 6782–6789 37 Kim, M.C., Kim, D.K., Lee, S.H., et al.: ‘Dynamic characteristics of superparamagnetic iron oxide nanoparticles in a viscous fluid under an external magnetic field’, IEEE Trans. Magn., 2006, 42, pp. 979–982 38 Furlani, E.P.: ‘Permanent magnet and electromechanical devices: materials, analysis and applications’ (Academic Press, New York, 2001) 39 Clime, L., Le Drogoff, B., Veres, T.: ‘Dynamics of superparamagnetic and ferromagnetic nano-objects in continuous-flow microfluidic devices’, IEEE Trans. Magn., 2007, 43, (6), 2007, pp. 2929–2931 40 Miltenyi, S., Muller, W., Weichel, W., Radbruch, A.: ‘High-gradient magnetic cell-separation with macs’, Cytometry, 1990, 11, pp. 231–238 41 Earhart, C.M., Nguyen, E.M., Wilson, R.J., Wang, Y.A., Wang, S.X.: ‘Designs for a microfabricated magnetic sifter’, IEEE Trans. Magn., 2009, 45, pp. 4884–4887 42 Furdui, V.I., Harrison, D.J.: ‘Immunomagnetic T cell capture from blood for PCR analysis using microfluidic systems’, LabChip, 2004, 4, pp. 614–618 43 Choi, J.W., Ahn, C.H., Bhansali, S., Henderson, H.T.: ‘A new magnetic bead-based, filterless bio-separator with planar electromagnet surfaces for integrated bio-detection systems’, Sensors and Actuators B, 2000, 68, pp. 34–39 44 Wunderlich, J.: ‘Cell-separation – methods and selected applications, Vol. 2 – pretlow, TG, pretlow, TP’, Anal. Biochem., 1984, 140, pp. 1–304 45 Berger, M., Castelino, J., Huang, R., Shah, M., Austin, R.H.: ‘Design of a microfabricated magnetic cell separator’, Electrophoresis, 2001, 22, pp. 3883–3892 46 Le Drogoff, B., Clime, L., Veres, T.: ‘The influence of magnetic carrier size on the performance of microfluidic integrated microelectromagnetic traps’, Microfluidics Nanofluidics, 2008, 5, pp. 373–381 47 Liu, Y.J., Guo, S.S., Zhang, Z.L., et al.: ‘Integration of minisolenoids in microfluidic device for magnetic bead-based immunoassays’, J. Appl. Phys., 2007, 102, pp. 084911–084911-6 48 Espy, M.A., Sandin, H., Carr, C., Hanson, C.J., Ward, M.D., Kraus, R. H.: ‘An instrument for sorting of magnetic microparticles in a magnetic field gradient’, Cytometry A, 2006, 69A, pp. 1132–1142 49 Sabourin, D., Snakenborg, D., Dufva, M.: ‘Interconnection blocks: a method for providing reusable, rapid, multiple, aligned and planar microfluidic interconnections’, J. Micromech. Microengng., 2009, 19, pp. 035021–035021 50 Ismagilov, R.F., Stroock, A.D., Kenis, P.J.A., Whitesides, G., Stone, H. A.: ‘Experimental and theoretical scaling laws for transverse diffusive broadening in two-phase laminar flows in microchannels’, Appl. Phys. Lett., 2000, 76, pp. 2376–2378 51 Ismagilov, R.F., Rosmarin, D., Kenis, P.J.A., et al.: ‘Pressure-driven laminar flow in tangential microchannels: an elastomeric microfluidic switch’, Anal. Chem., 2001, 73, pp. 4682–4687 52 Munir, A., Wang, J.L., Li, Z.H., Zhou, H.S.: ‘Numerical analysis of a magnetic nanoparticle-enhanced microfluidic surface-based bioassay’, Microfluidics Nanofluidics, 2010, 8, pp. 641–652
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ISSN 1751-8741
Experimental investigation of magnetically actuated separation using tangential microfluidic channels and magnetic nanoparticles Ahsan Munir1, Zanzan Zhu1, Jianlong Wang 2, Hong Susan Zhou1 1
Department of Chemical Engineering, Worcester Polytechnic Institute, 100 Institute Road, Worcester, MA 01609, USA College of Food Science and Engineering, Northwest A&F University, 28 Xinong Road, Yangling, Shaanxi 712100, People’s Republic of China E-mail: [email protected]
2
Abstract: A novel continuous switching/separation scheme of magnetic nanoparticles (MNPs) in a sub-microlitre fluid volume surrounded by neodymium permanent magnet is studied in this work using tangential microfluidic channels. Polydimethylsiloxane tangential microchannels are fabricated using a novel micromoulding technique that can be done without a clean room and at much lower cost and time. Negligible switching of MNPs is seen in the absence of magnetic field, whereas 90% of switching is observed in the presence of magnetic field. The flow rate of MNPs solution had dramatic impact on separation performance. An optimum value of the flow rate is found that resulted in providing effective MNP separation at much faster rate. Separation performance is also investigated for a mixture containing non-magnetic polystyrene particles and MNPs. It is found that MNPs preferentially moved from lower microchannel to upper microchannel resulting in efficient separation. The proof-of-concept experiments performed in this work demonstrates that microfluidic bioseparation can be efficiently achieved using functionalised MNPs, together with tangential microchannels, appropriate magnetic field strength and optimum flow rates. This work verifies that a simple low-cost magnetic switching scheme can be potentially of great utility for the separation and detection of biomolecules in microfluidic lab-on-a-chip systems.
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Introduction
Biomolecular separation using microfluidics technology involves capture, isolation and release of target biomolecules from impure samples. It is used for purification, pre-concentration and detection of biomolecules [1, 2], proteins [3, 4], DNA [5, 6] and cells [7, 8] in clinical diagnostics, drug discovery and microbiology. There are a number of techniques currently available on microfluidic platforms for particle separation, such as electrophoresis [9, 10], dielectrophoresis [11], size-based separation [12–14], pinched flow fractionation [15, 16], acoustic separation [17], inertial separation [18, 19] and magnetic bioseparation [8, 20–25]. Among these techniques, magnetically actuated methods seem to be more promising because of the simplicity of design and ease of operation. This method utilises surface-functionalised magnetic particles to trap target biomolecules through specific chemical binding followed by separation using magnetic manipulation. Magnetic bioseparation technique is dependent on the interaction of chemical bonds and therefore allows highly specific and selective biomolecular separation when compared with other techniques that rely on geometrical or physical properties of the species.
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Most of the magnetic bioseparation systems developed on microfluidic platform are based on magnetic micro-sized particles [2, 8, 26–28], however, less intensively studied microfluidic bioseparation [29–33] schemes are the emerging nanoscale magnetic particles. Compared with microparticles, nanoscale materials possess better properties that can be advantageously deployed in microfluidic devices. Higher surface to volume ratio [34] makes magnetic nanoparticles (MNPs) nearly ideal for the manipulation and detection of attached biomolecules [30, 35]. For example, it has been shown that functionalised MNPs have enhanced the detection of small molecules using surface plasmon resonance (SPR) spectroscopy [36]. The small size of MNPs causes minimal disturbance to the attached biomolecules [34] as well as provides enhanced interaction for chemical binding and tagging. Most importantly, they are super-paramagnetic in nature [34], that is, their magnetisation without a magnetic field is zero. The super-paramagnetic nature ensures that they stay suspended in carrier liquid when the magnetic field is removed. Unlike micrometre-sized particles, these particles do not irreversibly agglomerate or precipitate and can be repeatedly used subjected to magnetic fields of varying strength without causing any adverse effects. This also makes it easy for the removal or capture of tagged biomolecules of
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www.ietdl.org interest once the magnetic field is removed. Overall, the inclusion of MNPs in microfluidic devices for biomolecule separation, manipulation and detection will not only enhance the device functionality and separation performance but also broaden the utility of these devices in real-world applications. Separation of biomolecules using magnetic field actuation can be achieved either in conventional batch process or in continuous flow mode [37–40]. In the batch process [24, 41, 42], the magnetic particles tagged with target biomolecules are trapped or retained using magnetic field and subsequently released, after the removal of non-targets. A number of devices have been developed with various magnet designs [1, 28, 43] to accomplish magnetic bioseparation. Useful batch mode operation suffers from low separation efficiency, longer incubation and handling time, and results in significant contamination due to non-specific binding of impurities with magnetic beads [44]. More importantly, their incorporation in point-of-care microfluidic testing devices will require more complicated multi-step fluidic handling. On the other hand, continuous flow magnetic bioseparation processes overcome the above limitations because they employ magnetic fractionation, that is, continuous accumulative deflection of magnetic particles tagged to biomolecules. This method does not require multistep fluidic handling; moreover, higher magnetic field is also not a requisite because the process only depends on deflection rather than on complete trapping of MNPs. Continuous flow magnetic bioseparation can be distinguished into two types: one in which electromagnets or magnetic microstrips typically of alloy or ferromagnetic materials are integrated on the device substrate to generate a magnetic field gradient that deflects magnetic beads [22, 25, 45–47]. Substantial cost and effort is required to design and fabricate these systems. Alternatively, in the second type, a simple external permanent magnet assembly is used that provides greater flexibility and simplicity in device design to achieve higher magnetic field-assisted bioseparation [48]. The development of continuous flow magnetic bioseparation scheme together with a simple low-cost approach for selective injection or removal of biomolecules bound to MNPs is highly desirable to complement the existing microfluidic technology available for bioseparation. In this work, a simple, low-cost and generic microfluidic platform is assembled to demonstrate continuous flow magnetic bioseparation using tangential microfluidic channels and MNPs. A major innovation of this set-up lies in the fabrication of tangential microchannel that acts as magnetic microfluidic switch to manipulate flows containing MNPs and can accomplish efficient bimolecular separation. Standard moulding process combined with a novel rapid prototyping method is used in this work to develop low-cost polydimethylsiloxane (PDMS) microchannel. The fabrication method used in this work circumvents the requirement for a clean room [49]. It also eliminates the combination of two pieces of element, such as in standard fabrication method where negative or positive stamp on PDMS is combined with glass or silicon using plasma. This further overcomes the issue of leakage in microfluidic channels. Continuous switching/separation of MNP in a sub-microlitre fluid volume surrounded by neodymium permanent magnet are studied. On the basis of MNPs concentration measurement using optical technique, separation efficiency is analysed for scenarios with and without magnetic field. Separation performance of the set-up is also studied for a mixture containing non-magnetic IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 102–110 doi: 10.1049/iet-nbt.2012.0023
polystyrene (PS) particles and MNPs. The effect of flow rate on continuous flow separation of MNPs in tangential microchannel is also investigated. This work demonstrates that a simple low-cost magnetic switching scheme using tangential microchannel together with MNPs can be potentially of great utility, which can further improve the functional performance of magneto-fluidic bioseparation systems.
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Experimental materials and methods Microchannel fabrication
Low-cost rapid micromoulding technique was used to develop 75 mm long and 800 µm diameter tangential microfluidic channels. The steps used in fabricating the microchannels are shown in Fig. 1. The first step in preparing these channels involved the development of mould, which was prepared using aluminium wires each of 800 µm diameters. The wires were placed at the centre and approximately at half the depth of the empty Petri dish such that they overlap each other at an angle of 30°. For that, small holes were drilled using the stainless steel syringe, which was later closed using adhesive tapes. It was also made sure that they touch each other approximately at the centre. PDMS (Sylgard 184, Dow Corning, USA) with a base and curing agent mixed in a ratio of 10:1 was poured onto the mould and was degassed to remove any bubbles using desiccators. The uncured PDMS was baked in an oven (65°C) for 1 h. The final step was to peel off the cured-PDMS containing the aluminium wires from the Petri dish. The sides of the cured PDMS were cut using a razor blade, leaving a significant amount of the wire exposed outside. With the help of pliers the wires were carefully removed. To make this process easier, the cured PDMS block containing the embedded wires was washed with acetone, which swelled the PDMS and expanded the channels prior to pulling out the wires. An internal access area was created at the centre of overlap where the wires touched each other making the only connection between two microchannels. The microchannels were then connected with the tygon tubing using the stainless tip obtained from microsyringe. The tip was inserted into the microchannels to make leakage-free connection. 2.2
Microfluidic system set-up
A simple experimental set-up to carry out magnetohydrodynamic experiments is shown in Fig. 2. As shown in the figure, the microfluidic channels are connected with inlet and outlet via flexible tygon tubing. Lower microchannel inlet (LT1) is used to transport MNP solution, whereas deionised (DI) water flows from the upper channel inlet (UT1). To provide leak-free connections, a microsyringe tips made of stainless steel are embedded into the microchannel inlet/outlet for secure connections between the flexible tubes and the microfluidic chip. A differential pressure drop is maintained inside the channels by connecting the outlet of the microchannels (LT2 and UT2) to peristaltic micropump (P625 Peristaltic Pump, Instech, USA) using an in-house developed PDMS T-shaped connector. The flow rates were varied using a precise bi-directional speed controller on the pump. This simple method allows for a good control of the flow in the channels in suction mode. In order to have more precise control over the flow, individual syringe pumps can be used 103
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Fig. 1 Fabrication step used in developing leak-proof microfluidic channels
Fig. 2 Experimental set-up of magnetic field based separation using tangential microchannels and MNPs Inset shows the close-up of tangential microchannel with neodymium magnet
for each microchannel instead of peristaltic micropump. The pumps can be calibrated in order to take into account a small imbalance in the flow resistances of the two channels. An upward magnetic pull force is obtained using the permanent neodymium magnets (Grade N52, KJ Magnetics, USA), as shown in Fig. 2 (inset), near the access hole where the upper microchannel is connected to the lower microchannel. The permanent magnet was 1 × 1 inch in dimensions with 3/8-inch thickness. The ‘residual flux density’ of this magnet was 14 800 Gauss with magnetisation along the thickness of the magnet. Optical images of the bioseparation experiments at different time points were obtained using the digital camera (Sony Cyber Shot DSC-W530, Sony Electronics Inc., USA). MNPs of 200 nm diameter (fluidMAG-ARA Chemicell GMBH, Germany) with a concentration of 1 mg/ml were transported through lower inlet. The MNPs consisted of an inner core made up of magnetite (Fe3O4) crystals of approximately 12 nm diameter, which were embedded in a biocompatible 104 & The Institution of Engineering and Technology 2014
polysaccharide matrix for better stability that also prevented biodegradation. The overall diameter of the nanoparticles was approximately 200 nm, whereas the volume fraction of magnetite within a composite particle is 80%. For different flow rates, the effluent was collected at the outlets once all the solution has passed through the microchannels. The volume collected at the outlet was regularly verified to confirm the equal flow rates in both the microchannels. The concentration of MNP solution in the effluent was estimated from in-house determined calibration curve. The calibration curves were generated from original stock of MNPs solution diluted at different concentrations. A dynamic slight scattering instrument Zetasizer Nano S (Malvern Zetasizer Nano S, UK) was used. The Zetasizer Nano S measures the intensity of scattered light of various concentrations of sample at one angle; this is compared with the scattering produced from a standard one (i.e. Toluene). In general, Zetasizer is used to measure the size of molecules based on the count rate, which is a fairly IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 102–110 doi: 10.1049/iet-nbt.2012.0023
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Fig. 3 Calibration curve obtained for MNPs using scattering intensity obtained from Zetasizer Nano S Inset shows different concentrations of MNPs used in generating calibration curve
stable value of the sample calculated from dynamic scattering. This value is used as a method of determining the relative concentration of a sample of stable size – as the count rate goes down, so does the concentration. While the Zetasizer software does not automatically estimates sample concentration, the count rate is used in this work to calculate concentration of samples. Power-law calibration curve of scattering intensity (kilocounts per second, kcps) against concentration of MNPs (mg/ml) were obtained for 200 nm particles (R 2 = 0.9908) (see Fig. 3). To obtain the concentration of MNPs coming out of the system under various condition of flow rate, the outlet samples from the effluent was taken in a cuvette and placed in the Zetasizer to obtain the unknown scattering intensity (kcps) of the sample. The calibration curve was used to convert the scattering intensity into concentration (mg/ml). Since the inlet concentration of MNPs was known, percentage separated was calculated.
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whereas at higher aspect ratio the fluid exchange is minimal and the fluid largely continues through the intersection within the same fluid stream. To minimise the exchange of fluid between microchannels, we tested many microchannels and found that microchannel with a diameter of 800 µm produced best results. The Reynolds number for the flow rate (0.6–11 µl/s) used in the experiments ranges from 0.239 to 4.42. For such a low Reynolds number, the inertial term in the Navier–Stokes equation can be neglected. This type of flow is referred as Stokes flow or Creeping flow and occurs in systems with small flow length scales. Based on the Reynolds number computation and experimental observation, the flow profile was always creeping instead of parabolic, and thus, the flow within the microchannels was always remained laminar. To investigate the transport of MNP solution in microchannels, the movement of the nanoparticles between the two tangential streams in the absence of any applied magnetic force is examined. The flow rates of the two channels were the same (∼5 µl/s). A 100 µl of MNP solution having a concentration of 1 mg/ml were injected in the lower microchannel, whereas DI-water flow was maintained in the upper microchannel. The transport of MNPs was recorded after every 2 s. It can be seen from Fig. 4 that after 6 s almost all the solution continues through the intersection within the same flow stream with a very small amount of solution transferring to upper stream because of diffusion across the interface.
Results and discussion
3.1 Qualitative and quantitative analysis of MNPs separation Magnetic manipulation and switching of nanoparticles between two flow streams is a complementary way of separating biomolecules or cells in microfluidic devices when these biomolecules are tagged with nanoparticles. It is based on the attraction of the nanoparticles tagged biomolecules to regions with higher magnetic field intensity. In this section, qualitative analysis of the switching or separation of MNPs is performed with the aid of imaging using a digital camera. The set-up of the experiment is already described in Section 2. In order to examine the MNP movement within the magnetic fields, tangential PDMS microchannel was fabricated using a simple and inexpensive benchtop fabrication method, as described earlier. It has been shown by Ismagilov et al. [50, 51] that the flow fields in tangential microchannels are independent of the contact area but strongly depends on the channel aspect ratio. A too low aspect ratio results in divergence of fluid from one channel into another channel, IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 102–110 doi: 10.1049/iet-nbt.2012.0023
Fig. 4 Flow of MNPs in the tangential microchannel in the absence of magnetic field, where 100 µl of MNP solution having a concentration of 1 mg/ml was injected in the lower microchannel 105
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www.ietdl.org In the next experiments, the movement of MNPs was investigated in the presence of magnetic field force. A neodymium magnet (N52) was aligned on top of the intersection of the two microchannels such that the edge of the magnet is very close to the intersection in order to provide maximum magnetic field force. The assembly of magnet is shown in Fig. 1 and described in Section 1.2 in more detail. It was found that the distance between the intersection of the two microchannels and the edge of the neodymium magnet was approximately 2 mm. The magnetic field intensity was computed using our in-house
developed COMSOL™ numerical code [21, 52], which were also validated using the well-developed analytical expressions given by Furlani et al. [38]. The magnetic flux density at this distance was computed to be in the range of 0.6–0.8 T. Mass transfer of 100 µl of MNP solution having a concentration of 1 mg/ml entering the lower microchannel was recorded after every 2 s. It can be seen from Fig. 5a that after 6 s MNPs preferentially move upwards because of the magnetic force acting on them near the intersection and flows with the upward stream. A significant amount of MNPs switching was achieved near the intersection that can
Fig. 5 MNPs switching between microfluidic channels using Neodymium magnet a Snapshot of tangential microchannel at different times. MNPs were injected in the lower microchannel b Close-up of tangential microchannel after 18 s shows MNPs switching 106 & The Institution of Engineering and Technology 2014
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www.ietdl.org be seen in Fig. 5b. A very small quantity of MNPs still emerges from lower stream, which could be due to moderate disturbance in flow field arising from imperfect channel geometry which causes roll up of some particles. In order to further investigate the performance of switching of MNPs, Zetasizer experiments were performed to evaluate the concentration of MNPs exiting both the outlets. The sample from the outlets is collected until all the solution has passed through the microchannel. It is taken in a cuvette and placed in the Zetasizer to obtain unknown scattering intensity (kcps) of the sample. Previously determined calibration curve is used to convert the scattering intensity into unknown outlet concentration (mg/ml). Each experiment both in the presence and absence of permanent magnet was performed in triplicates and the average values together with the standard deviation were reported. A 100 µl of MNP solution having a concentration of 1 mg/ml was injected into the lower microchannel with a flow rate of 5 µl/s. It can be seen from Fig. 5 that when the magnetic field is not deployed, most of the MNP follows the same flow stream with the concentration of MNPs exiting the lower microchanel was approximately 0.88 mg/ml. The concentration of MNPs found in the upper microchannel was very small ( ∼ 0.064 mg/ml approximately). This proves that no switching or separation takes place in the absence of magnetic field. It was also found that around 5.5% MNPs were not found either in the upper or lower microchannel. This could be due to the fact that some of the MNPs got trapped within the microchannel and never exited the system. Some of this error could also arise from instrumental error because of the correlation made between scattering intensity and concentration. However, when the magnetic field was used, as seen from data given in Fig. 6, 0.90 mg/ml of the MNPs were switched from lower microchannel to upper microchannel. This was because these MNPs experience magnetic pull force near the area of intersection and were transferred into upward flow stream. A very small amount ( ∼ 0.067 mg/ml) was found in the lower microchannel with approximately 3% not found either in the upper or lower microchannel. It was seen that out of 3% some of these MNPs got trapped on the inner walls of microchannel because of strong magnetic force. From the above analysis,
Fig. 6 Concentration of MNPs eluted from upper and lower microchannel when 100 µl of 1 mg/ml MNPs were injected through lower microchannel at flow rate of 5 µl/s in the absence and presence of magnetic field (Neodymium magnet, N52) IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 102–110 doi: 10.1049/iet-nbt.2012.0023
it can be demonstrated that 90% separation of MNPs was achieved using tangential microfluidic channels. 3.2
Effect of flow rate on MNPs separation
In this section, the effect of flow rate on the MNPs separation in tangential microchannel is investigated. Variation in solvent flow rates impacts the residence time of MNPs within the microchannels as well as the drag and magnetic forces acting on these particles. Although the magnetic force is independent of the flow rate, but the time that the magnetic force acts on the particles is related to the average residence time of the particles in the vicinity of the magnet. A longer residence time means smaller flow rates that translate to smaller drag forces; therefore if the particles are exposed to magnetic force for longer time, there will be a strong tendency that these particles will experience higher magnetic force than drag force and as a consequence will be pulled across the interface and into the other microchannel. To test the role of flow velocity on magnetic switching or separation, a series of experiments were performed using a constant magnetic field and varied flow rates. MNPs with an initial concentration of 1 mg/ml and volume of 100 µl were injected from the lower microchannel at varying flow rates. The concentration of MNPs that exited the intersection in the upper and lower microchannels was detected using Zetasizer instrument together with the procedure described in Section 1.1. Each experiments both in the presence and absence of permanent magnet was performed in triplicates and the average values together with the standard deviation were reported. Since the inlet concentration of MNPs was known, percentages of MNPs separated between two tangential microchannels were calculated. It can be seen from Fig. 7a, that switching of MNPs takes place when magnetic field is used. A higher percentage of MNPs were pulled from the lower microchannel to upper microchannel at lower flow velocity ( < 5 µl/s). However, when the flow velocity was increased beyond 5 µl/s, a linear decrease in MNP switching was observed. This is because at lower flow velocity, the drag force acting on the MNPs was small and the magnetic force acted for greater amount of time on the MNPs near the junction. To investigate quantitatively, the average residence time of MNPs at the junction region ( ∼ 800 µm) was calculated for different flow rates. It can be seen from Fig. 7b that at lower flow rates, the magnetic force acts for greater amount of time on MNPs ( ∼ 3000 ms) when they are passing the junction as compared with higher flow rates. This result in more number of MNPs pulled in the upper flow stream from lower microchannel, which was not the case when the flow rate was increased. Another interesting observation was made in the absence of magnetic field when the flow rates were smaller than 5 µl/s. It was found that at a very low flow rates ( ∼ 0.6 µl/s), small amount of MNPs ( ∼ 18%) were pulled in the upper channel. In order to test whether this behaviour was due to diffusional transport, an estimate for the diffusion coefficient was obtained using the Stokes–Einstein equation for the diffusivity given in (1). DMNP =
kB T 6phRMNP
(1)
where RMNP is the radius of the MNP that is diffusing, η is the viscosity of the suspending fluid (water), kB is the Boltzmann 107
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Fig. 7 Effect of flow rate on MNPs separation a Percentage of MNPs eluting from lower (O) and upper (Δ) microchannel at different flow rates in the presence and absence of magnetic field. MNPs initial concentration was 1 mg/ml and 100 µl of the sample was injected from lower microchannel b Residence time of MNPs in the junction region against flow rates. Error bar represent the standard deviation obtained after three injections
constant and T is the temperature. This formula is applicable for a spherical particle much larger than the solvent molecules. In this work, we assume MNPs as spherical particles suspended in water. Based on the above equation, the diffusion coefficient for 200 nm MNPs was calculated to be 2.14 × 10 − 12 m2/s. In order to further investigate, the Peclet number, Pe, which is the product of system length and fluid velocity, divided by diffusivity was computed. It was found that for all flow conditions, Pe was much greater than one implying that diffusion is not dominant and small migration of MNPs could be because of convective flow field that diverted near the imperfect junction boundaries. This trend was minimised as the flow rate was increased. A more thorough investigation will be needed to account for such observed behaviour, which is beyond the scope of this work. From these experiments it was illustrated that the application of magnetic field causes the MNPs to move from the lower microchannel to upper microchannel. However, the percentage of MNPs that can be magnetically as well as non-magnetically transported into the upper microchannel is dramatically influenced by the flow rate of both fluid streams. As the flow rate decreases from 11 to 0.6 µl/s under magnetic field, the percentage of MNPs eluting from the upper microchannel increases from 48.8 to 93.4% with more drastic change found in between flow rates of 5–11 µl/s. The improved separation achieved at lower flow rates was not simply a result of the increased residence time but is a function of both drag and magnetic force acting on these MNPs. By carefully calibrating the fluid flow, an optimum value of flow rate can be achieved to provide maximum switching of MNPs together with higher throughput essential for bioseparation application. The sizes of MNPs also play an important role in the separation process. In this work, only one type of MNPs was used. The magnetic force acting on MNPs is directly 108 & The Institution of Engineering and Technology 2014
proportional to size of MNPs; a larger diameter MNPs will result in higher magnetic forces, therefore the flow rate and strength of magnets can be adjusted to avoid capturing of particles on MNPs or even clogging microchannels. A more detailed optimisation study taking into account MNPs size, flow velocity, junction length and magnetic field strength is the next step before realising the advantage of using this concept. 3.3 Magnetic separation of a mixture of magnetic and non-magnetic particles Magnetically actuated switching of biomolecules tagged with MNPs from one fluid stream into another, while leaving behind non-magnetic particles, is an excellent strategy to achieve microfluidic-based separation of biomolecules continuously. This strategy was demonstrated and tested in this section. A mixture of 1 mg/ml of PS (60 nm) and 1 mg/ ml of MNPs (200 nm) with a 1:1 volume ratio was injected from the lower microchannel in the presence of magnetic field. The total volume of the mixture was 100 µl and the injection rate of 5 µl/s was maintained in both the upper and lower microchannels. Samples from the outlets were collected and analysed using Zetasizer. Both the size as well as concentration measurement was performed using the instrument. Figs. 8a and b give the histogram of the size distribution of the particles eluting from the upper and lower channels. It can be seen from the results that the average size of particles eluting from the upper and lower channels were around 273.1 and 68.75 nm, respectively. This illustrate that most of the MNPs were pulled into the upper microchannel because of magnetic force leaving behind non-magnetic PS particles in the lower microchannel. A slight error in size estimation could be due to presence of MNPs in lower and PS in upper IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 102–110 doi: 10.1049/iet-nbt.2012.0023
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Fig. 8 5 µl/s
Magnetic field-based separation of a mixture of PS (60 nm) and MNPs (200 nm) injected from lower microchannel at a flow rate of
a Average diameter of sample eluted from the upper microchannel b Average diameter of sample eluted from the lower microchannel c Percentage of MNPs eluted from the upper and lower microchannels d Percentage of PS eluted from the upper and lower microchannels
microchannel. Based on the particle size histograms, it is evident that the application of the magnetic field preferentially removed the MNPs from the lower microchannel. In order to investigate the separation or switching efficiency, the concentrations of MNPs and PS were computed using the method described in earlier sections. It can be seen from Figs. 8c and d that 89.9% of MNPs were pulled into the upper microchannel, whereas 90.6% of PS stayed in lower microchannel. From the above analysis, it is evident that magnetic field-assisted preferential switching of MNPs can be efficiently utilised in separating biomolecules in microfluidic devices.
4
Conclusion
Magnetic fields can be effectively used to manipulate the movement of MNPs together with biomolecules attached on their surfaces. In this work a continuous switching/ separation of MNPs in a sub-microlitre fluid volume surrounded by neodymium permanent magnet is studied. A simple, low-cost and generic microfluidic platform is developed for proof-of-concept experimentation. On the basis of MNPs concentration measurement, the movement of the nanoparticles between the two tangential streams in the absence and presence of the applied magnetic force was investigated. It was found that negligible switching of MNPs takes place in the absence of magnetic field, whereas 90% of switching was observed when the magnetic field was employed. The flow rate of MNP solution had a dramatic impact on separation performance. A too high flow rate resulted in decrease in switching of MNPs, IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 102–110 doi: 10.1049/iet-nbt.2012.0023
whereas a too low flow rate did not significantly improve the separation efficiency. It was observed that by carefully calibrating the fluid flow, an optimum value of flow rate can be found to provide maximum switching of MNPs together with higher throughput essential for the bioseparation application. Separation performance was also studied for a mixture containing non-magnetic PS particles and MNPs. It was found that MNPs preferentially moved from lower microchannel to upper microchannel, resulting in an efficient separation from non-magnetic particles. The proof-of-concept experiments performed in this work further demonstrates that microfluidic-based separation of biomolecules can be efficiently achieved using functionalised MNPs, together with tangential microchannels, appropriate magnetic field strength and optimum flow rates. This work further demonstrates that a simple low-cost magnetic switching scheme can be potentially of great utility for the separation and detection of biomolecules and cells in lab-on-a-chip systems.
5
Acknowledgments
The authors gratefully acknowledge the support from the National Science Foundation (grant no. CMMI-1030289) and National Natural Science Foundation of China (grant no. 31101274)
6
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