Building bio-assays with magnetic particles on a digital microfluidic platform.

New Biotechnology Volume 00, Number 00 April 2015

REVIEW

Review

Building bio-assays with magnetic particles on a digital microfluidic platform Tadej Kokalja,b,1, Elena Pe´rez-Ruiza,1 and Jeroen Lammertyna a b

Department of Biosystems—MeBioS-Biosensor Group, KU Leuven, Leuven, Belgium Institute of Metals and Technology, Ljubljana, Slovenia

Abstract

Digital microfluidics (DMF) has emerged as a promising liquid handling technology for a variety of applications, demonstrating great potential both in terms of miniaturization and automation. DMF is based on the manipulation of discrete, independently controllable liquid droplets, which makes it highly reconfigurable and reprogrammable. One of its most exclusive advantages, compared to microchannel-based microfluidics, is its ability to precisely handle solid nano- and microsized objects, such as magnetic particles. Magnetic particles have become very popular in the last decade, since their high surface-to-volume ratio and the possibility to magnetically separate them from the matrix make them perfect suitable as a solid support for bio-assay development. The potential of magnetic particles in DMF-based bio-assays has been demonstrated for various applications. In this review we discuss the latest developments of magnetic particle-based DMF bio-assays with the aim to present, identify and analyze the trends in the field. We also discuss the state-of-the art of device integration, current status of commercialization and issues that still need to be addressed. With this paper we intend to stimulate researchers to exploit and unveil the potential of these exciting tools, which will shape the future of modern biochemistry, microbiology and biomedical diagnostics. Contents Magnetic microparticles on DMF platforms . . DNA-based applications. . . . . . . . . . . . . . . . . DNA amplification . . . . . . . . . . . . . . . . . . . . DNA sequencing . . . . . . . . . . . . . . . . . . . . . . Single nucleotide polymorphism genotyping . Immunoassays . . . . . . . . . . . . . . . . . . . . . . . Cell-based applications . . . . . . . . . . . . . . . . . Digital assays . . . . . . . . . . . . . . . . . . . . . . . . Sample preparation on chip . . . . . . . . . . . . . Integrated DMF platforms . . . . . . . . . . . . . . .

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Corresponding author. Tel.: +32 16 32 14 59; fax: +32 16 322955. Lammertyn, J. ([email protected]) 1

Both authors contributed equally.

http://dx.doi.org/10.1016/j.nbt.2015.03.007 1871-6784/ß 2015 Elsevier B.V. All rights reserved.

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Please cite this article in press as: Kokalj, T. et al., Building bio-assays with magnetic particles on a digital microfluidic platform, New Biotechnol. (2015), http://dx.doi.org/10.1016/ j.nbt.2015.03.007

1

NBT-772; No of Pages 19 REVIEW

Commercialization . . . . . Conclusions and outlook. Acknowledgements . . . . . References. . . . . . . . . . . .


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Introduction

Review

The microfluidics field emerged in the 1990s when it was recognized as a promising alternative for liquid handling due to its inherent properties including handling of tiny fluid volumes with high precision and reduced time and cost to perform the analysis [1,2]. Since then it has developed rapidly and became widely available due to new technologies (e.g. soft lithography, pneumatically-actuated valves) which decreased the price and facilitated the fabrication of microfluidic devices [3]. Microfluidics has been used for a wide variety of applications, though the bio-medical research field seems to be the most attractive and most relevant target for both basic research and further commercialization [4–7]. One of the first demonstrated microfluidics approaches was the continuous flow microfluidics (CFM). The fluid in CFM is constrained by microchannels and all of the fluid manipulations are performed in continuous non-segmented flows. Although CFM paved the way for a fundamentally new manner of manipulating fluids on the micro level, it has several drawbacks [1]. First, when scaling-up the number of reactions on the platform, the size and complexity of the device quickly increases. Second, even more critical is the low flexibility of the microfluidic devices, since every new application or even slight modification of the established protocol requires a new design and fabrication. Therefore, most of the CFM devices are highly specialized. A solution to the scale-up issues of CFM was found in droplet-based microfluidics [1]. In this case small liquid plugs that are immersed in an immiscible continuous phase (gas or liquid) within enclosed microchannels are employed. Droplet-based microfluidic systems provide many advantages for high-throughput screening applications as thousands of droplets can be generated per second. However, its weakness is the limited interaction with individual droplets, since the droplets are forced to follow the flow patterns given by the predefined microchannel structures. Moreover, there is always some ‘‘dead volume’’ of sample in channel restricted systems that cannot be used for analysis, this is especially critical when the overall sample volume is already small [1].

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An alternative droplet-based microfluidic approach that does not use predefined microchannels and allows for small sample volumes is digital microfluidics (DMF) [8–10]. DMF is based on the manipulation of discrete, independently controllable fluid droplets, which makes it highly reconfigurable and reprogrammable. The droplet actuation is performed on a flat hydrophobic surface which is usually covered by an additional plate in a sandwich device. As opposed to the channel-restricted microfluidics in which pumps and valves are actuated to control the flow in the channel, DMF uses a unique actuation force to address individual droplets. In DMF platforms electrowetting-on-dielectric (EWOD) [11,12] is by far the most popular mechanism for droplet manipulation (Fig. 1), although some other droplet actuation mechanisms have been demonstrated, including magnetic actuation [13,14], surface acoustic waves [15], optoelectrowetting [16], mechanical actuation [17]. The basic droplet operations, such as dispensing, transport, splitting, merging and mixing allows for highly complex protocols which can be executed in parallel and in a programed manner. This, joined to the advantageous flexible system architecture and high-fault tolerance capability [18], makes this platform appealing for building true lab-on-a-chip (LOC) devices. An extra advantage that DMF offers over the channel-based microfluidics is its exceptional capability to handle solid particles, such as magnetic beads. Transporting and handling these particles in microchannel-based microfluidics can be challenging, since they tend to clog the channels. This is especially significant, seeing that in the late years magnetic particles have been proven as multifunctional manipulation vehicles in LOC systems. Because of their large specific surface for chemical binding and the ability to manipulate them by magnetic fields, the use of magnetic particles is growing, especially in bio-applications [19–21]. The purpose of this review is to highlight the importance of magnetic particles as a useful tool for building bio-assays on a DMF platform. We first give an overview of magnetic particle properties and the different strategies for their manipulation in DMF platforms in order to achieve the basic droplet operations such as

FIG. 1

(A) Schematic representation of the basic mechanism of EWOD actuation: the electric potential on the electrode changes the contact angle and therefore surface wettability. Consequently the droplet moves in the direction of increased wettability. (B) A droplet encapsulated in an oil shell on an electrode array is waiting for the EWOD actuation. (C) Comparison of size of a one Euro coin and a digital EWOD-based DMF chip. 2

www.elsevier.com/locate/nbt Please cite this article in press as: Kokalj, T. et al., Building bio-assays with magnetic particles on a digital microfluidic platform, New Biotechnol. (2015), http://dx.doi.org/10.1016/ j.nbt.2015.03.007

Ref.

Year

Authors

Application

Droplet actuation

Type/size of magnetic particles

Purpose of MP

Magnetic particle manipulation

Type of platform

Detection

Media

[37]

2010

Hua et al.

RT-PCR

EWOD

Superparamagnetic

2006

Lehmann et al.

Magnetic

Ferromagnetic 3 mm

Fluorescence

2013

Chiou et al.

Magnetic

Not specified

Fluorescence

Oil

[13]

2007

Pipper et al.

RT-PCR

Magnetic

Superparamagnetic 100 nm Superparamagnetic 2–5 mm Superparamagnetic

Double plate, Integrated platform One plate, open system One plate, open system One plate, open system One plate, open system One plate, open system Double plate

Hexadecane, oil Oil

[29]

DNA purification RT-PCR

Integrated, permanent magnet Electromagnetic coils

Fluorescence

[23]

DNA capture, preconcentration, washing Droplet transportation, DNA capture, DNA purification Droplet transportation DNA capture, DNA purification Droplet transportation RNA capture, RNA purification Droplet transportation

Fluorescence

Air

–

Oil

Fluorescence

Air

Fluorescence

Air

Double plate, integrated platform

Luminiscence

Oil

External, permanent magnet

Double plate

Fluorescence

Oil


Double plate

–

Air


Double plate

–

Air

External, permanent magnet External, permanent magnet Integrated, permanent magnet External, permanent magnet External, permanent magnet External, permanent magnet Integrated, permanent magnet External, permanent magnet Electromagnetic coils

Double plate

Fluorescence

Not specified

Double plate

Chemiluminiscence

Oil

Double plate, integrated platform Double plate

Chemiluminiscence Fluorescence

Hexadecane, oil Air

Double plate

Chemiluminiscence

Air

Double plate

Electrochemical

Air

Double plate, integrated platform Double plate

Chemiluminiscence

Air

Fluorescence

Air

One plate, open system Double plate

Hall Sensor

Air

Fluorescence

Air

[14] [26]

2007

Welch et al.

DNA sequencing

EWOD

Superparamagnetic 2.8 mm

[46]

2011

Kim et al.

DNA sequencing

EWOD

Superparamagnetic

[47]

2013

Kim et al.

DNA sequencing

EWOD

Superparamagnetic

[49]

2012

Shen et al.

SNP detection

EWOD

[52]

2008

Sista et al.

Immunoassay

EWOD

[53]

2008

Sista et al.

Immunoassay

EWOD

[54]

2011

Vergauwe et al.

Immunoassay

EWOD

[55]

2012

Ng et al.

Immunoassay

EWOD

Superparamagnetic 2.8 mm Superparamagnetic 1 mm Superparamagnetic 1 mm Superparamagnetic 15 nm Superparamagnetic

[56]

2014

Shamsi et al.

Immunoassay

EWOD

Superparamagnetic

[57]

2013

Choi et al.

Immunoassay

EWOD

Superparamagnetic

[34]

2014

Vergauwe et al.

Immunoassay

EWOD

Ferromagnetic 3 mm

[58]

2013

Bhalla et al.

Immunoassay

EWOD

Not specified

[66]

2010

Shah et al.

Cell-based assay

EWOD

Superparamagnetic

Capture cells

Boles et al.

C2CA

Magnetic EWOD

DNA sequencing

EWOD

Superparamagnetic, 1 mm Superparamagnetic, 2.8 mm

External, permanent External, permanent External, permanent External, permanent Integrated, permanent

magnet magnet magnet magnet magnet


Review

REVIEW

2011

2011

Ku¨hnemund et al.

RT-PCR

Magnetic

[45]

[44]

2014

Zhang et al.

PCR

Droplet transportation, DNA extraction Capture ligation complex, separation, washing DNA immobilization for sequencing; separation, washing DNA immobilization for sequencing; separation, washing DNA immobilization for buffer exchange, washing, size selection DNA immobilization for buffer exchange, washing, size selection Capture DNA, preconcentration Capture antigen, separation, washing Capture antigen, separation, washing Capture antigen, preconcentration Capture antigen, separation, washing Capture antigen, separation, washing Capture antigen, separation, washing Capture antigen, separation, washing Label

[40]

2013

Ohashi et al.

Electromagnetic coils

NBT-772; No of Pages 19

Summary of the reviewed articles classified with respect to the bio-application, the mechanism employed for droplet actuation, the type and size of the used magnetic particles, the role of the magnetic particles, the means of magnetic manipulation, the type of DMF platform, the detection strategy and the media of the assay.


www.elsevier.com/locate/nbt 3 Please cite this article in press as: Kokalj, T. et al., Building bio-assays with magnetic particles on a digital microfluidic platform, New Biotechnol. (2015), http://dx.doi.org/10.1016/ j.nbt.2015.03.007

TABLE 1


Oil

Air

–

Fluorescence

Fluorescence

Double plate, integrated platform

Double plate, integrated platform Double plate, integrated platform Sample preconcentration, DNA extraction Capture non-specific proteins, sample preparation 2012

2014

[73]

[74]

Mei et al.

Protein depletion

EWOD

Superparamagnetic 1 mm Superparamagnetic 10 mm EWOD

EWOD

RNA extraction and purification qPCR 2014 [72]

Jebrail et al.

EWOD Digital assay 2013

Delattre et al.

Oil

[71]

Witters et al.

Integrated, permanent magnet Integrated, permanent magnet

Fluorescent imaging Double plate magnet

magnet

Fluorescent imaging Double plate magnet

Isolation and confinement of single target molecules Isolation and confinement of single DNA molecules Sample preconcentration, RNA purification EWOD 2013 [70]

Witters et al.

2014 [67]

Tewari Kumar et al.

Cell-based assay Digital assay

EWOD

Superparamagnetic 2.8 mm Superparamagnetic 2.8 mm Superparamagnetic 2.8 mm Superparamagnetic

Immobilization of cells

External, permanent External, permanent External, permanent Integrated, permanent

magnet

Oil

Fluorescent imaging Double plate

Air

Detection Type of platform Authors Year Ref.

TABLE 1 (Continued )

Application

Droplet actuation

Type/size of magnetic particles

Purpose of MP

Magnetic particle manipulation

Review 4

Air


Media

REVIEW

droplet transport, mixing, splitting and most importantly separation. Then we present the most significant and recent bio-applications of magnetic particles on DMF platforms, including DNA purification, amplification and sequencing, immunoassays, cellbased assays, digital assays and finally integrated solutions ready for clinical testing. The main properties of the selected reviewed papers are summarized in Table 1. For each system there is information on the bio-application, the droplet actuation mechanism, the type and size of magnetic particles, the role of magnetic particles, the means of magnetic particle manipulation, the type of DMF platform, the detection strategy and the media employed for the assay. Therefore, Table 1 gives the most condensed overview of the ‘‘state of the art’’ and current trends in the field and is referred throughout the review for comparison and discussion. Finally, we conclude the paper with a summary and outlook of the future challenges of the field.

Magnetic microparticles on DMF platforms Magnetic particles typically consist of a magnetic core and a nonmagnetic shell or an ensemble of nanometer-sized magnetic grains embedded in a non-magnetic matrix. The magnetic material content and distribution inside the bead determines its magnetic properties, while biocompatibility and stability in different media are determined by the selection of the shell/matrix material. The magnetic material is generally an iron-oxide (Fe3O4) because of its high saturation magnetization and the non-magnetic matrix is often a polymer such as polystyrene. For their application in bioassays the particle surface needs to be functionalized in order to allow covalent bonding or adsorption of biomolecules. The first, most important property that makes magnetic microparticles very attractive for use in bio-assays is their large surface-tovolume ratio which increases available area for chemical binding or adsorption and, consequently, the analyte/target capture efficiency. The second property is their ability to be magnetized under the influence of an external magnetic field. The magnetization depends on both the particle material properties and the magnitude of the external magnetic field. Upon the termination of the magnetic field the particles might stay magnetized (ferromagnetic particles) or not (superparamagnetic particles). The magnetic force can be higher on the ferromagnetic particles, however they can get clustered due to their residual magnetization, even after removal of the external magnetic field. In contrast, superparamagnetic particles get easily re-suspended and maintain their high surface-to-volume ratio. The superparamagnetic particles are far more popular for the bio-application as can be seen in Table 1. However the selection of the proper magnetic particles is application-specific and should therefore be carefully considered depending on the target application in order to get the optimal performance of the system. More about the selection of suitable magnetic particles can be found in extended reviews by Gijs et al. [19,20]. Droplet movement by magnetic actuation of suspended particles can be achieved by the movement of an external permanent magnet [22] or by activation of embedded electromagnets (coils) on the surface [23] (Fig. 2A). Systematic investigation of droplet movement, coalescence and splitting on an open hydrophobic surface was done by Long et al. [24]. The authors used a permanent magnet and analyzed the behavior of the droplet at various magnet velocities and magnetic particle loadings. By varying these



FIG. 3

Basic droplet operations on a DMF platform: dispensing, translation, mixing and splitting. Adapted and reprinted with permission from Ref. [9]. Copyright Annual Reviews 2012. FIG. 2

(A) Magnetic particles can be used to induce droplet movement by magnetic actuation through application of an external magnetic field. (B) Magnetic particles are only present to facilitate the separation step. In this last case there are two possible magnetic separation strategies: (C) The droplet is pinned to the surface and the magnetic particles pellet is removed by means of a moving magnet, or (D) the magnetic particles pellet is pinned to the surface and the droplet is actuated so particles are left behind.

two parameters they realized three distinct operating regimes; steady droplet transport, particle extraction, and magnet disengagement. Droplet transport occurs at low magnet speeds, while particle extraction requires a higher speed and a higher loading. Magnet disengagement occurs at high velocities and lower particle loadings. The authors provide a simple force balance model and discuss the effects of the particle type, the droplet size, the surrounding oil layer, the surface tension and the viscosity. Most of the contemporary digital microfluidics systems do not use magnetic particles for droplet manipulation, but rather use methods based on electrostatic actuation, like EWOD. In this case the magnetic particles are only used to facilitate the separation and purification assay steps (Fig. 2B). In EWOD the droplet is actuated on an extremely hydrophobic dielectric layer deposited onto the actuation electrodes as presented in Fig. 1. When no electric field is applied to the electrode, the droplet will rest on the surface with a large contact angle (low wettability). By applying an electric field, the dielectric and droplet are polarized and the attractive force virtually increases the wettability of the surface. Therefore, through activation of the electrodes it is possible to control the displacement of the droplet (Fig. 3). In EWOD-based systems actuation the magnetic particles are widely used as a solid carriers which can be extracted from the droplet by means of magnetic fields. This is especially useful in bio-assays for performing steps like extraction and washing. Since the separation and the concentration of the target are essential steps in bio-microfluidic applications, they are also the most common application of magnetic particles on DMF platforms. While one external force exerted on the liquid droplet is sufficient to achieve droplet transport, fusion and mixing, two different forces are needed to achieve particle separation from the liquid droplet. There are two basic strategies for effective

separation: (i) the droplet is pinned to the surface while the pellet of magnetic particles is removed (Fig. 2C) or (ii) the pellet of magnetic particles is pinned to the surface and the droplet is moved away (Fig. 2D). Various methods of droplet pinning were described in the literature, i.e.: hydrophobic patches, confined chambers, surface topography. Lehmann et al. [25] presented a method in which droplets were immobilized on a surface by hydrophilization while magnetic beads were manipulated by means of actuation of electric coils. Magnetic beads washing was successfully demonstrated in this work (Fig. 4A). Similar to Lehmann’s approach is the application of ‘‘surface energy traps’’ (SETs) presented by Zhang et al. [26] in which a permanent magnet was used to move the magnetic particle pellets. SETs are areas of high surface energy on a glass substrate coated with a low surface energy Teflon1-AF film. By using them, authors have demonstrated a full range of magnetic operations on a magnetically actuated droplet platform, including droplet transport, fusion, particle extraction and dispensing. Shikida et al. [27] have introduced confined chambers for different reactants to physically retain the droplets while transferring the magnetic bead cluster from one chamber to another through connecting channels. Similarly, Zhang et al. [28] and Chiou et al. [29] have demonstrated the surface topographyassisted droplet manipulation, where they introduce a microstructure on the surface to retain the droplet but not the microparticles (Fig. 4B). A significantly different method of particle separation was presented by Shah and Kim [30]. They used a EWOD platform to pin the droplets and create a liquid neck or a ‘‘fluidic conduit’’ between a sample droplet and a buffer droplet (Fig. 4C). The long fluidic path minimized the diffusion and fluidic mixing between the droplets and allowed for high collection efficiency (99%) and elimination of non-desired species in just one purification step. However, the creation of the thin fluidic neck between the droplets is highly dynamically instable and therefore challenging. The alternative to droplet pinning is the immobilization of the magnetic particle pellet, normally with the help of an external magnet. The droplet is then removed by another actuation mechanism (i.e. EWOD). Efficient in-droplet separation on an EWOD platform was demonstrated by Wang et al. [31]. Particle separation


Review




Review FIG. 4

Different concepts of magnetic particle separation on a DMF platform. (A) Particle actuation by electromagnetic microcoils on the surface. The liquid is retained by a hydrophilic patch. Reprinted with permission from Ref. [25]. Copyright Elsevier B.V. 2006. (B) Examples of geometry assisted separation. The droplet is retained by obstacles on the surface and the magnetic beads are moved by means of a moving permanent magnet. Reprinted with permission from Ref. [28]. Copyright The Royal Society of Chemistry. (C) A ‘‘Fluidic conduit’’ is formed between the droplets by means of electrowetting forces and the magnetic particles migrate under the influence of magnetic field. Reprinted with permission from Ref. [30]. Copyright The Royal Society of Chemistry 2009. (D) Combination of EWOD and a permanent magnet. The magnetic particles are retained at a desired location by a permanent magnet, while the liquid droplet is removed by electrowetting forces. Reprinted with permission from Ref. [32]. Copyright Springer-Verlag 2007.

in this case was done by means of a permanent magnet and electrowetting forces. After one splitting step they achieved high separation efficiency (91%) of magnetic beads, but 35% of nonmagnetic beads were not separated. With an additional droplet merging and splitting step they achieved 94% and 92% separation efficiency respectively for magnetic and nonmagnetic beads. Fouillet et al. [32] also immobilized the magnetic beads by means of a permanent magnet, while the surrounding solution was exchanged with new solution in consecutive steps for efficient washing (Fig. 4D). Other basic droplet operations were discussed in 6

their work such as; dispensing, translation and merging. The authors reported very high magnetic beads retention efficiency (99.8%), exceeding significantly earlier experiments with magnetic beads in which the retention efficiency was relatively low and some of the beads were lost in the washing buffer. In order to improve washing efficiency on a dry surface Shah et al. [33] proposed a ‘‘meniscus assisted’’ highly efficient magnetic collection and separation on EWOD platform. They discuss the importance of the interfacial forces on magnetic beads. In dry environment the particles can adhere to the surface and they



can be swept back into the droplet if the droplet is moved on the surface. They demonstrate the increase of particle collection efficiency, from 73% to 99%, after using ‘‘meniscus-assisted’’ microbeads collection. The influence of actuation voltage and magnetic particles concentration on particle separation was studied by Vergauwe et al. [34]. They did a force balance analysis for a EWOD—static permanent magnet system and found three possible operation regimes for droplets: (i) normal droplet transport, (ii) droplet transport is blocked, (iii) particles are extracted. At low particle concentrations the droplet transport was always achieved, however, at increased particle concentration the magnetic force may prevail over electrowetting force and the droplet cannot move upon activation. In that case the actuation voltage must be increased correspondingly. Beside magnetic particle separation, magnetic particle mixing is also an important issue in all microfluidic systems, including DMF platforms. Vergauwe et al. [34] proposed two different magnetic particle-based stirring strategies: In the first one, they created internal flows in a droplet by shuttling it back and forth above a permanent magnet and in the second one they took advantage of self-assembled chains of ferromagnetic particles that are formed under application of an external magnetic field. Authors proved that when the applied magnetic field was rotating, the chains were broken, forming smaller clusters of particles that were turning around and could be used to stir the droplet. With this strategy effective mixing was achieved. They reported a significant improvement (90%) in sensitivity, compared to passive mixing based on diffusion, and an overall decrease of variability (3% in coefficient of variation). The effect of magnetic particles mixing was also demonstrated by Chiou et al. [29] on food dye mixing. In their work, active mixing of the magnetic particles resulted in a homogenous solution after 2 s, while the diffusive process barely showed any mixing after 50 s. The authors applied the developed mixing procedure in the demonstration of a PCR protocol (see Section 3). Although several different magnetic actuation and separation strategies have been demonstrated and presented in the literature, EWOD actuation in combination with permanent magnets constitutes the most popular approach for handling magnetic particles in DMF systems, especially for bio-applications, as it will be shown in the following sections.

DNA-based applications DNA amplification Polymerase chain reaction (PCR) has been established as a standard technique to selectively and exponentially amplify trace amounts of DNA. It has revolutionized the field of modern biology and has become a routine tool in life science research. In recent years, the concept of miniaturizing and automating PCR systems through microfluidics has achieved great attention, because of the potential to drastically improve the speed and performance, besides offering portability and cost effectiveness. In 2003, Pollack et al. [35] described the first DMF platform towards PCR applications. However, amplification was performed on an external conventional real-time PCR system and only the actuation of droplets containing standard PCR reagents was tested on a EWOD-based DMF platform. Three years later the first integrated EWOD-based DMF/PCR system was reported by Chang et al. [36]. The authors implemented an on-chip temperature control system, consisting

REVIEW

of microheaters and a micro temperature sensor, allowing for precise thermal cycling required for the PCR amplification. In order to demonstrate the performance of the integrated platform, a detection gene for Dengue II virus was successfully amplified and detected on-chip. Later, in 2010, Hua et al. [37], also from Pollack’s lab, presented the first automated EWOD-DMF platform for multiplexed real-time PCR assays. In contrast to than traditional PCR, in which results are collected after the reaction is complete, real-time PCR measures amplification as it occurs. The amplified DNA is labelled with fluorescent dyes and fluorescence is measured during each thermal cycle, therefore requiring the integration of a detection system. The authors designed a platform consisting of two different parts: (1) an instrument including all the required control and detection features (i.e. power supply, control electronics, fluorimeter module, heaters and cartridge deck including magnets) and (2) a disposable microfluidic cartridge (see Section 8). To evaluate its performance, authors demonstrated amplification of genomic DNA. Remarkable amplification efficiency (94.7%) was achieved and the equivalent of a single genome was detected for a methicillin-resistant S. aureus model. The reproducibility was comparable to conventional bench-top real-time PCR instruments; however DNA amplification from actual clinical samples was only possible when magnetic particles, were introduced in the system. Magnetic particles allowed sample concentration and solution exchange (i.e. washing) on chip, which made their device suitable for the detection of DNA pathogens present in low concentrations in real samples. As a proof of concept they extracted and purified microbial DNA from spiked whole blood samples and concentrated it from a sample volume of 5–10 mL into a droplet of 660 nL; subsequently, amplification was successfully performed. As can be seen in Table 1 magnetic actuation is a predominant alternative to EWOD actuation in DMF platforms for PCR applications. In this case magnetic particles usually have the dual role of biomolecule carriers and actuation vehicles. In 2006, Lehmann et al. [23] presented the first droplet manipulation system in which magnetic particles are employed both as force mediators and substrates for biomolecule capture. The actuation method was based on a switchable matrix of electromagnetic coils integrated on the surface. In order to achieve droplet splitting and magnetic separation a hydrophilic/hydrophobic pattern was fabricated on the surface of the chip. As a proof of concept, authors performed DNA extraction and purification on chip, but did not accomplish DNA amplification. This was done by Chiou et al. [29], who used the same actuation concept to perform all processing steps from genomic DNA extraction from whole blood to real-time PCR on a single cartridge. In this case separation of magnetic particles and droplet splitting was achieved by means of topographical barriers and the chip cartridge was interfaced with thermal cycling and optical detection modules. The authors also tested the effect of magnetic particle agitation and mixing on the DNA capture and elution processes, and demonstrated that the amount of DNA was substantially enhanced in comparison to a diffusion-dependent method. Two other groups (Pipper et al. [13] and Ohashi et al. [14]) also employed magnetic actuation in DMF systems for PCR applications, but in their work actuation was done by an external permanent magnet. On one hand, the platform presented by Ohashi et al. consisted of a tray reactor with a hydrophobic bottom


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Review FIG. 5

Integrated platform for multiplexed genetic detection on a SETs-enabled magnetic droplet platform. (A) Chip layout with buffers predeposited on the specific locations. (B-F) Different assay steps: (B) mixing of crude sample with lysis buffer and SET-assisted particle washing and extraction, (C) SET-assisted aliquoting of the eluent, (D) Mixing with PCR mixture, (E) SET-assisted extraction of the magnetic particles from the reaction mixture and (F) Samples ready for PCR amplification. Reprinted with permission from Ref. [26]. Copyright WILEY-VCH Verlag GmbH & Co. 2013.

surface, which was filled with oil. Aqueous droplets containing magnetic microparticles were applied into the oil solution and a magnet was placed beneath, to attract the particles. When the magnet was moved the particle-containing droplet followed its movement. Authors performed highly efficient DNA amplification by moving a droplet containing PCR reaction mixtures over two regions at different temperatures. On the other hand, Pipper et al. reported a chip consisting on a hydrophobic flat perfluorinated surface on which droplets of aqueous-in-oil suspensions of magnetic particles were deposited. Similarly, a motor-controlled permanent magnet was used to actuate the droplets. In order to perform RT-PCR the chip was placed on a microfabricated heater with an integrated optical detection system. A crucial difference is that whereas Ohashi et al. employed particles purely for assisting droplet actuation, here magnetic particles were also used as solid surface for capturing the RNA. In order to achieve particle separation, the concentration of particles was raised up to 2500% with 8

respect to the benchtop protocol to overcome surface tension resistance. With this platform authors detected avian influenza virus (H5N1) directly from a throat swab in less than 30 min. The RNA was automatically isolated, purified and preconcentrated on the surface of the magnetic particles and then released and subjected to RT-PCR amplification. In the same line, Zhang et al. [26] performed multiplexed realtime PCR starting from blood samples employing the previously described SETs-based platform, which also relied on magneticbased droplet actuation (Fig. 5). In their platform DNA was isolated by magnetic particle-based solid phase extraction. As a proof of concept, authors performed multiplexed detection of three different biomarker genes with this platform. It is important to notice that all platforms based on magnetic actuation are open, one-plate systems, contrary to EWOD-based platforms, which are typically double-plated devices. The reason is that, even though EWODbased DMF chips can be either designed as single or double-plate



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FIG. 6

Schematics of a C2CA amplification protocol on a DMF chip. (A) DMF chip and assay layout in which positions of droplets with reaction mixes are depicted. Mixing of two droplets is illustrated with green arrows with double arrowheads. Mixing of single droplets is illustrated by green arrows with single arrowhead. Magnetic particle transfer between droplets is indicated with red arrows. (B) Flow chart illustrating magnetic particle extraction and transfer and droplet mixing in consecutive assay steps. Reprinted with permission from Ref. [40]. Copyright The Royal Society of Chemistry 2014.

devices, single-plate EWOD chips are not capable of dispensing and splitting droplets [38], two operations crucial for bioassay implementation on-chip. Besides PCR, several isothermal DNA amplification techniques have been investigated as an alternative. Among them, rolling circle amplification (RCA) [39] has become popular due to its simplicity, flexibility and robustness. Specificity and sensitivity of this technique can be improved by using circularized padlock probes as a template, since circularizing of a padlock probe is strictly a target-dependent action relying on two different hybridization events. Also, by including an additional amplification step that consists of monomerization of the concatemer, recirculation of the monomers and generation of new RCA products. This is the so-called circle-to-circle amplification (C2CA), which allows for a billion fold or greater DNA amplification. However, it consists of many steps which make integration a difficult task and only few studies have been reported in which microfluidic chips are used for ¨ hnemund the detection of RCA amplified molecules. Recently, Ku et al. [40] integrated for the first time C2CA on a digital microfluidic platform (Fig. 6), with all the steps integrated on chip except for the heating. In their work, magnetic particles are used as a solid phase for the bioassay, increasing the sensitivity and speed by allowing separation of circles from unreacted padlock probes and sample remains. The authors developed an extraction and transfer protocol for 1 mm sized superparamagnetic particles

on the DMF platform. In the described platform a permanent neodymium magnet was placed above the top plate in proximity of the particle containing droplet, and the influence of the distance between the magnet and the particles was investigated by changing the thickness of the top plate and the position of the magnet edge above the electrodes. Both parameters were found to play an important role in the particle extraction. Thin top plates of maximum 0.7 mm thickness and positioning the magnet on the border between two consecutive electrodes, instead in the middle of them, gave the best results. With these conditions 1 mm sized magnetic particles with a concentration as low as 1.6 105 particles/mL were extracted successfully and bigger particles of 2.8 mm diameter could be extracted in 3 104 particles/mL concentration. In the overall assay, which consisted of three washing steps, particle extraction efficiency was 99.5%. With these optimized conditions, C2CA was performed on chip and a 1 aM concentration of P. aruginosa DNA was detected. Moreover, the assay efficiency was proven to be the same as the one obtained in off-chip control tubes.

DNA sequencing One of the main applications of DNA amplification is the preparation of target DNA for sequencing. DNA sequencing is one of the most important techniques for the study of biological systems, especially since the revolution of next-generation sequencing



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(NGS) technologies in the past decade [41]. The first NGS technology was pyrosequencing, developed by 454 Life Sciences [42] and George Church’s lab [43]. It is based on the detection of released pyrophosphate (PPi) during DNA synthesis. In a cascade of enzymatic reactions, PPi is converted to adenosine triphospate (ATP) and finally visible light is generated with an intensity proportional to the number of nucleotides added to the strand. The main part of the overall cost of pyrosequencing is the amount of costly reagents (i.e. enzymes) needed. Thus, the use of smaller devices, such as microfluidic platforms, offers a miniaturized and cost-effective alternative and several research groups have tried to adapt pyrosequencing to a microfluidics format. Within microfluidics, the droplet-based format is better suited since the pyrosequencing process is essentially a series of repeated liquid handling steps. In 2011, two different groups (Boles et al. [44] and Welch et al. [45]) proved the feasibility of using EWOD-DMF to pyrosequence DNA. In both cases, authors implemented a three-enzyme pyrosequencing protocol in which enzymes, deoxyribonucleotide triphosphates (dNTPs) and DNA templates were contained in individual droplets. In the described three-enzyme pyrosequencing reaction, complete removal of excess nucleotides at each step is a major determinant of read lengths. Therefore, the DNA templates need to be thoroughly washed between nucleotide additions. Both groups adopted a similar strategy, exploiting the potential of magnetic particles, in order to perform these washing steps. They captured the DNA template on magnetic particles and anchored them onto a specific chip location with the help of permanent magnets, while repeatedly adding fresh buffer droplets. Subsequently the magnetic particles were resuspended by transporting the droplet away from the magnet. In this way, the functionalized beads could be thoroughly rinsed and also reagents could be added and excess split away without losing DNA. Boles et al. designed a custom bench-top instrument with integrated stationary permanent magnets in which a microfluidic chip cartridge was placed. Their template DNA was a 229 bp fragment from Candida parapsilosis genomic DNA, amplified by PCR and then purified. Signal detection was performed by a photo multiplier tube (PMT) also integrated in the deck of the instrument. They performed sequencing using two different protocols: a ‘‘de novo’’ protocol, in which nucleotides are added in a repeated cyclical way, and a ‘‘resequencing’’ protocol, in which nucleotides are added in a predetermined, non-repetitive order that anticipates the actual sequence of the template. The last one generated over 60 bp of sequence with 100% accuracy. Although the overall sequencing throughput was limited, their work tested the suitability of DMF platforms for low-cost DNA sequencing. Alternatively, Welch et al., focused on studying the optimal magnet geometries for the formation of localized magnetic regions that allowed controlling the concentration of magnetic beads in the dispensed droplet, since they worked with picoliter droplets and at this small scale variations in the bead content per droplet can have a significant effect on the recorded signal. This is important, because the template DNA is immobilized on the magnetic particles, and therefore the concentration of these particles is proportional to the concentration of DNA used for pyrosequencing. A three-magnet geometry, consisting of two paired triangular magnets juxtaposed at 1208 with respect to one another and a third magnet separated by 50 mm and oriented toward its two sister components, gave the optimal field gradient to create the desired localized magnetic 10


region. In this work, authors did not perform full DNA sequencing, but as a proof of concept of the whole pyrosequencing process, a calibration curve was created by varying the concentration of ATP in the substrate solution. The limit of detection was 7 nM ATP, which is lower to the equivalent change in signal that would be observed upon addition of a single nucleotide. Although both mentioned works successfully implemented sequencing protocols on DMF chips, preparation of the DNA for subsequent sequencing was performed off-chip. This shortcoming was compensated by Patel and co-workers, who integrated and automated on a DMF platform the preparation of genomic DNA for following NGS (i.e. DNA fragmentation, washing, amplification and size selection) by combining continuous-flow and droplet-based manipulations on the same device [46,47]. The presented platform merges several sample preparation modules with a DMF chip by a capillary interface. The sample preparation modules consist of a network of tubes connected to a valve manifold which is attached to a syringe pump. For DNA fragmentation and PCR amplification the sample tubing network was guided through channels drilled in a custom-machined thermal block, whereas for DNA washing and size selection it was routed above a permanent magnet. The interface comprises small capillary cylinders swaged into the side ports of the DMF chip and connected to the end of the tubing net. In this platform magnetic particles are first employed for buffer exchange and washing steps between DNA fragmentation and PCR amplification and later for DNA size selection. In order to validate the developed platform, authors prepared NGS libraries from both human and genomic DNA samples on-chip as used them in a NGS sequencer achieving more than 99% of alignment with respect to the reference genome. However, in this platform the extraction of genomic DNA from whole biological samples is still performed off-chip.

Single nucleotide polymorphism genotyping SNPs are inherited single base DNA variations at a defined genetic location, which are found in at least 1% of the human population [48]. Most genetic differences between humans are SNPs, making them suitable as markers for diseases and different responses to drug treatments. DNA sequencing, which was described in the previous section, is the gold standard approach for SNP detection. But in the recent years, SNP genotyping, which consist on screening of individuals to detect already known SNPs, is becoming popular due to the rise of new assays to detect genetic mutations which offer a simpler, faster and more cost-effective alternative with potential for integration in point-of-care (POC) devices. In 2013, Shen et al. [49] implemented SNP detection on a temperature-controllable EWOD-DMF platform for the first time. In their work, magnetic particles were used as a carrier to capture and purify the target DNA and SNP detection was performed by an enzyme-based ligation assay. In this assay the target DNA is partially complementary to both a capture probe and a detection probe, which is usually modified with a label. The ligase enzyme is only able to connect the capture probe to the detection probe when the target sequence is perfectly paired with them. If a mismatch is present due to a SNP mutation in the target sequence, the ligase is not able to link both sequences (capture and detection) and thus complete hybridization with the target sequence is not possible. Afterwards a thermal step allows denaturation of only the


NBT-772; No of Pages 19 New Biotechnology Volume 00, Number 00 April 2015

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mismatched sequences. These enzyme-based ligation assays have been first described by Landegren and coworkers [50] and ensure high assay specificity thanks to the combination of short oligonucleotide hybridization and selective enzymatic activity. Since thermal washing of the mutant probes is a crucial step of the assay, it is also critical to implement controlled heating capabilities on-chip, thus Shen et al. integrated a microheater on the DMF platform by micropatterning the surface of the top plate of the chip. The microheater temperature was controlled by an infrared scope. In their system capture DNA oligos were bound to the surface of the magnetic particles, which simplified washing steps on-chip and allowed for target concentration with the help of an external magnet. Detection probes were labelled with a quantum dot and detection was done by placing the chip under a fluorescence microscope. With this platform, authors successfully distinguished a SNP on a 30 bp DNA probe.

Immunoassays Immunoassays take advantage of the high affinity and specificity in binding between an antigen and its homologous antibodies to detect and quantify biomarkers. Among all immunoassay formats, heterogeneous immunoassays, in which the antibody-antigen complex is immobilized on a solid surface and unbound molecules are washed away, are the most popular due to their higher sensitivity [51]. In order to miniaturize and automate them, microfluidics appears as a promising platform because these assays are inherently rate-limited by the mass transport of biomolecules to the solid surface. They can benefit from the reduction of diffusion length and incubation times which are inherent to microfluidics. Microparticles offer a valuable alternative as solid support for heterogeneous immunoassay implementation on microfluidic platforms, since they offer a large active surface area for biomolecule binding, while their functionalization can be decoupled from the microfabrication process. All available examples of immunoassays on DMF platforms are based on the use of magnetic particles and exploit their unique advantages for enabling antigen capture and the subsequent washing steps. The first magnetic particle-based immunoassay implemented on a DMF chip was developed by Pamula and coworkers in 2008 [52]. In their work (Fig. 7), a solution containing a mixture of magnetic particles, antibodies and blocking proteins was first prepared off-chip, and then dispensed and mixed with a droplet of the antigen solution in order to form the antibody-antigen complexes. Subsequently, the beads were immobilized using an external magnet and unbound reagents were washed from magnetic particles, by a serial dilution approach. Next the particles were re-suspended and detection was performed. The authors evaluated various parameters that affect the attraction of magnetic beads, such as the buffer solution, the magnetic pulling force and the position of the magnet on the chip. They found that adding a small percentage of surfactant to the buffer and using a magnet with a pulling force of 5.5 N placed underneath the droplet, were the best conditions for an efficient and quick separation. Authors also focused on attaining efficient washing of the beads while maintaining 100% retention, which they achieved by a serial dilution-based protocol, consisting of three steps that were repeated until sufficient dilution was accomplished. The three steps were: (i) immobilization of magnetic particles by an external

FIG. 7

Example of a protocol for performing heterogeneous immunoassays on a DMF platform developed by Pamula’s group. (A) dispensing of reagents, (B) incubation, (C) immobilization of magnetic beads, (D) removal of supernatant and washing, (E) adding fresh buffer. Reprinted with permission from Ref. [52]. Copyright The Royal Society of Chemistry 2008.

magnet, (ii) dilution of the droplet containing the particle suspension by merging with a droplet of fresh buffer and (iii) splitting the droplet to remove excess solution from the particles. In order to test their platform, non-competitive immunoassays for insulin and interleukin-6 were successfully performed on chip. The same authors integrated all the operations to perform a completely automated magnetic bead-based heterogeneous immunoassay on physiological samples on a DMF platform and demonstrated an immunoassay for cardio troponin I (cTn1) on whole blood [53]. In essence the implemented immunoassay was the same but in this case they also compared the performance of ‘‘on-magnet’’ incubation, this means moving the bead-containing droplet back and forth above the magnet, and ‘‘off-magnet’’ incubation when the magnet was first removed. They showed that the ‘‘off-magnet’’ incubation strategy considerably reduced the assay time. They also designed a handheld device including all control electronics for autonomous operation in which the DMF chip can be integrated (see Section 8). In 2011, Vergauwe et al. [54] developed an immunoassay for IgE protein on a DMF platform. The assay strategy was similar to the one followed by Pamula et al., but in this work the detection antibody was replaced by a DNA aptamer. They were able to detect as low as a 150 nM concentration of IgE on-chip. This is one of the few reported works that employs superparamagnetic nanoparticles instead of microparticles (see Table 1). Authors hypothesized that nanoparticles, unlike microparticles, have a unique colloidal nature by which their charge and their Brownian motion cause the dispersion to be stable and thus they are homogeneously distributed through the droplets even during dispensing. They reported




Review FIG. 8

Comparison of magnetic particle washing protocols for magnetic bead-based DMF immunoassays. (A) Serial dilution washing protocol, in which the droplet containing the particle suspension is first diluted by merging with a droplet of fresh buffer. (B) Supernatant separation washing protocol, in which particles are first focused into a pellet that remains behind when the supernatant droplet is actuated away. Reprinted with permission from Ref. [55]. Copyright American Chemical Society 2012.

average variability of 2.83% in the nanoparticle concentration of the dispensed droplets compared to 30% previously reported by Fouillet et al. [32] using 10 mm particles. Above-mentioned works only described non-competitive immunoassays, while Wheeler and coworkers in 2012 reported the first competitive immunoassay on a DMF platform and described a new format for particle-based immunoassays [55,56]. Here, instead of the previously described dilution series-based protocol, authors employed a supernatant separation method for performing washing steps of magnetic particles and compared the performance of both strategies (Fig. 8). In the supernatant separation method, a magnet was placed under the particles containing-droplet so the particles focused into a pellet that remained behind when the supernatant droplet was actuated away. After separation from the supernatant, the immobilized particles were resuspended by removal of the external magnet plus addition of a fresh droplet over the pellet in order to form a new droplet that was then shuttled in a circular motion across four electrodes. This new strategy allowed for the complete separation of the supernatant dilution from the magnetic particles in a single step. When authors compared both approaches, the supernatant separation method seemed to be more efficient, because it minimized background signal to the same levels using significantly less washing steps. Using this strategy, they successfully developed a competitive immunoassay for estradiol, a natural estrogen in mammals, and a non-competitive immunoassay for thyroid-stimulating hormone (TSH), used to evaluate thyroid function. Both showed a limit of analytical performance acceptable for clinical screening. One year later, the same group presented an automated DMF platform capable of performing immunoassays from sample to analysis with minimal manual intervention [57] (see Section 8). Their platform, consisting of an interface for microfluidic control integrated with a magnetic lens assembly to focus the magnetic field from a permanent magnet and a detector, allowed the 12

performance of up to eight simultaneous digital microfluidic magnetic separations and was used to implement a full factorial design of experiments (DOE) optimization for the previously developed TSH non-competitive immunoassay. The authors varied the analyte concentration, the sample volume and the sample incubation time in order to come up with an optimized assay protocol. This was the first report of a DOE optimization for immunoassays in a microfluidics platform and with this approach, the detection limit and sample incubation time were reduced by 5fold and 2-fold, respectively, when compared with those from previous work. In a recent publication, Vergauwe et al. [34], also implemented the supernatant separation method to develop an immunoassay for IgG on an EWOD-based DMF platform, probing once again a strong increase in washing efficiency. In this case, the authors focused on improving the bioassay reaction dynamics by a particle-based stirring process to accelerate antigen capture (see Section 2). With this strategy the sensitivity of the IgG assay was increased with 90% and concentrations in the low ng/mL range were detected. As can be seen in Table 1, there is only one article reporting a magnetic particle-based immunoassay on a DMF platform that does not employ an external magnet for performing magnetic separation. An additional novelty of this platform, presented in 2013 by Bhalla et al. [58], is that magnetic particles are employed as a label for the analyte. The authors designed a cross-shape layout in which the central electrode was the measurement electrode which was coated with nitrocellulose in order to facilitate the binding of primary antibodies to its surface. Immunocomplexes were formed by subsequent incubation of this electrode with droplets containing the analyte and magnetic particles coated with the detection antibody, respectively. Afterwards, a magnetic washing step was performed in order to remove the unreacted beads from the electrode surface, since in this case were the magnetic beads that actually indicate the presence of analyte.



This magnetic washing was done by actuating the chip electrodes to act as an electromagnet so the unreacted beads experienced a magnetic repulsion force and only the beads linked to the formed immunocomplexes stayed on the electrode surface. The presence of those beads was measured by a Hall sensor. With this platform authors could perform an immunoassay in 7 min, although details about the outcome of the assay are not given in the paper. The implementation of immunoassays on digital microfluidic platforms inherently deals with the problem of biofouling, or unwanted adsorption of biomolecules, especially proteins, on the chip surface. This results in a loss of chip reliability and functionality. DMF chips are particularly susceptible because they are generally coated with a fluorinated polymer such as Teflon1-AF. Therefore, several strategies were reported to protect the surface of DMF devices of unwanted biofouling. Usually the chips are filled with a protective liquid such as silicone oil. This strategy is widely used, also in other applications such as DNA or cell-based assays (see Table 1) because it not only reduces the amount of surface adsorption, but also significantly decreases droplet evaporation rates. However, working in an oil media has some disasvantages as some on-chip functions can be incompatible with it. Also precautions need to be taken to prevent leaks. To circumvent these problems, in some of the reported works each of the individual droplets is encapsulated in a thin oil shell [49]. This allows the performance of the on-chip assays in air, but still avoids biomolecule contact with the chip surface. Alternatively, Wheeler and coworkers, proposed as a solution supplementing the protein-containing droplets with pluronic additives [55–57]. This approach, which also allows working in air, was previously introduced by the same authors [59,60] and proven to successfully prevent biofouling in both immuno- and cell-based applications.

Cell-based applications Cells are often studied as a living model for complex biological systems and cell-based assays constitute a versatile and powerful tool in biomedical research, since they can be designed to measure virtually any cellular or biochemical function. Cell-based assays are typically performed in well plates using microplate readers that are integrated in robotic analysis platforms to facilitate fluid handling. A major disadvantage of those platforms is their cost and complexity [61]. As a result, more applications of microfluidics to cell-based analysis have been investigated over the last years. DMF has been recently introduced as a promising alternative to perform miniaturized cell-based assays, offering several advantages when compared to channel-based microfluidics. Besides reduced reagent consumption and the absence of channel clogging issues, they feature low shear stress during liquid operation, which is particularly important when dealing with cells [62]. Barbulovic-Nad et al. [63] were the first to use DMF for cell manipulation and analysis. They studied the effects of EWOD manipulation on the cells vitality by comparing the viability and proliferation of actuated and non-actuated cells, and showed that no significant difference was observed. Later, research focused on concentration and isolation of cells on chip [64,65] and in this context magnetic particles appeared as a powerful tool to efficiently capture, isolate and concentrate the cells. Shah et al. [66], integrated for the first time magnetic particles on a DMF chip

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for cell-based applications. By merging and mixing two droplets containing, respectively, beads functionalized with specific antibodies and cell samples, they successfully isolated CD8+ lymphocytes from other peripheral blood components. The circulating flow inside the droplet led to excellent mixing and a high capture efficiency, over 92%. Recently, Tewari Kumar et al. [67] reported the isolation of plant protoplasts in suspension enabled by magnetic particles. The authors automated for the first time the osmotic treatment of a group of isolated single protoplasts by capturing and constraining them at a defined location with the help of an external applied magnetic field. First, the concentration of magnetic particles to efficiently capture protoplast was optimized and, once the protoplast was isolated, a water permeability test was performed on-chip, giving results in line to the ones found in literature. With the incorporation of magnetic particles, the authors overcame one of the major limitations for monitoring and tracking individually non-adherent cells on-chip, which consists on anchoring suspension cells on a fixed position of the chip.

Digital assays Optical single-molecule detection methods, also known as digital assays [68], have become valuable analytical techniques for a broad variety of bio-assays, enabling sensitive detection of relevant biomolecules down to the attomolar range. This ultrasensitive detection is possible by the isolation of individual target biomolecules into small reaction chambers where the signal is generated and detected. Recently, magnetic particles have been implemented to capture individual molecules and confine them into the discrete femtoliter sized wells, making digital assays simpler and more efficient. First, the biomolecules are captured on the particles surface. At very low target concentrations, the ratio of protein molecules to beads is very small and the percentage of beads that captures a target molecule follows a Poisson distribution which dictates that magnetic particles carry either one single target molecule or none. Target molecules are typically labeled with an enzyme that after addition of the substrate will generate a fluorescent signal. In this way the signal is generated and confined only in the wells that contain captured biomolecules and therefore by counting the number of fluorescent wells we can quantify individual molecules. Rissin et al. [69] were the first to exploit magnetic particles for digital assays and developing the first beadbased detection of single protein molecules. In their platform, centrifugal forces are employed to load individual magnetic particles into microwell arrays fabricated on an optical fiber bundle, where they are sealed with a silicone gasket in the presence of a droplet of the fluorogenic substrate. With this approach the authors developed a digital enzyme-linked immunosorbent assays (digital ELISAs) for prostate specific antigen (PSA) and tumor necrosis factor-a (TNF-a) in serum, achieving limits of detection in the low attomolar range in both cases. However, due to the nature of the bead loading process the distribution of single beads in the microwell array is arbitrary and loading efficiency is only about 60%. This implies that wells which contain a bead have to be identified beforehand in order to precisely determine the ratio of ‘‘on’’ and ‘‘off’’ wells for digital quantification. In 2013, Witters et al. [70] introduced EWOD-based DMF as a promising alternative for printing and sealing single magnetic beads into ultra-small


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Review FIG. 9

DMF-based seeding of single superparamagnetic beads for performing digital bio-assays developed by Witters et al. (A) Schematic representation of the DMFchip. The top plate features an array of 62,500 hydrophilic-in-hydrophobic microwells in which individual particles are trapped. (B) Schematic demonstration of how suspended beads in a droplet are attracted towards the array of microwells with the help of a magnet while the receding droplet meniscus removes excess beads off the surface. Reprinted with permission from Ref. [70]. Copyright The Royal Society of Chemistry 2013.

reaction wells (Fig. 9). In their platform, arrays containing more than 60,000 hydrophilic-in-hydrophobic, femtoliter-sized wells were patterned on the top plate of the DMF chip. By placing a permanent magnet above this array and transporting a droplet containing the magnetic particles over it, the particles are trapped on the wells with single bead resolution. With this approach authors achieved very high particle loading efficiencies of almost 100%, which potentially avoid the need of counting the wells that contain a magnetic particle making the assay truly digital. Moreover, DMF offers the possibility of integrating all fluid manipulations on chip, which allows the performance of miniaturized and fully automated single-molecule assays. As a proof of concept, authors quantified biotinylated protein b-galactosidase down to attomolar levels in the developed DMF platform and, to further validate it, they also demonstrated the on-chip detection of DNA with single-molecule resolution [71]. In order to do that, the authors captured target DNA on magnetic particles that are functionalized with specific capture DNA probes and subsequently label them with a detection probe linked to an enzyme. By seeding these functionalized beads on individual wells and sealing them in the presence of a fluorogenic substrate, single DNA molecules could be counted. With this approach they quantified DNA from bacteria Pseudomonas aeruginosa down to femtomolar levels.

Sample preparation on chip A lab-on-a-chip (LOC) is a miniaturized system that automates various laboratory functions within a single integrated platform. Based on this definition a DMF-LOC device would ideally integrate and automate all the necessary steps between sample acquisition and display of the results. However, as it has been shown in previous sections, most of the reported work only address a part of this entire process. Since sample volumes are often too large for direct treatment on a microfluidics platform and usually contain the analyte in very low concentration, one of the key challenges is the integration of sample preparation on-chip. This is a major issue due to the large discrepancy between volumes of real samples 14

(several mL to mL) and typical LOC operating volumes (few mL or sub-mL). However, some examples where authors addressed the challenge of bringing and handling ‘‘real-world’’ samples into DMF devices were recently described and in them, the magnetic particles played a key role. Jebrail et al. [72], from Patel’s group, designed a ‘‘World-to-DMF’’ interface in which an integrated unit (extraction module) repeatedly takes a large-volume sample through a 10 mL droplet region on the chip (purification module). Here the analyte is captured on magnetic particles that are subsequently concentrated with an external magnet (Fig. 10). To validate their platform, authors extracted RNA from human whole blood lysates (110–380 mL) and further purified it in microvolumes (5–15 mL) on the DMF chip. When compared to conventional RNA extraction and purification protocols, this platform was 2 times faster and consumed 12 times less reagents. Moreover it is highly reconfigurable and has potential for application to different analytes in different samples. Another example of DMF-based platform with integrated sample preparation was presented by Delattre et al. [73]. They designed an integrated device (30 cm 50 cm 40 cm) consisting of two modules (a sample preparation module and a detection module) and applied it to pathogen detection in order to perform biological environmental monitoring. The two modules were connected for complete protocol integration from sample preparation to DNA detection. In the sample preparation module pathogens were preconcentrated from few mL of complex samples, and afterwards pathogens DNA was extracted and purified into a few mL of buffer samples. For this authors employed a two-step protocol based on magnetic particles: in the first step pathogens are captured and preconcentrated and in the second step, after addition of the lysis buffer, DNA is captured in new beads surface. The detection module was a DMF-based PCR miniaturized platform. Transfer of the purified DNA extracts and PCR reagents from the first to the second module was done by a pump and a tube connecting a reservoir chamber with the DMF chip inlet. The platform was employed to quantify E. coli in both buffer samples and real



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FIG. 10

World-to-digital microfluidics (DMF) system presented by Jebrail et al. Detailed schematic representation of (A) the extraction module and (B) the interface between the extraction and purification modules. Reprinted with permission from Ref. [72]. Copyright American Chemical Society 2014.

samples obtained with a commercial air collection module. In the two cases results were comparable and purification yield was around 50%. An additional issue with ‘‘real-world’’ samples is that very low concentration of target molecules is present in a complex matrix. This is especially challenging in the case of physiological samples (i.e. blood), which are rich in highly abundant proteins (HAPs), such as immunoglobulins. Recently, Mei et al. [74] reported a method of protein depletion integrated in an EWOD-DMF set up. The method relies on magnetic particles coated with specific antibodies or proteins towards HAPs which were brought into contact with the sample solution. After active incubation with the sample by moving the droplet on the chip for several minutes (to enhance mixing), the beads were removed and the sample droplet was collected for further analysis. The authors evaluated the kinetics of on-chip depletion and showed than in 10 min they could achieved 95% of depletion efficiency, while current commercially available techniques, such as chromatography columns, require between 20 and 60 min to achieve similar efficiency. With this method, the need of high levels of sample dilution on-chip is eliminated.

Integrated DMF platforms An additional and important requirement for LOC devices, especially if they are aimed to point-of-care testing, is portability. Therefore, the DMF chip itself and the required additional instrumentation should be compact, robust and ideally inexpensive. Pamula’s group presented in 2008 the first DMF integrated platform for point of care testing [53]. They developed a handheld

prototype instrument (Fig. 11A) consisting of a touch screen, an USB interface, an integrated PMT detector and appropriate control electronics for autonomous operation. Disposable microfluidic cartridges (3 6 cm) were used with the instrument for analysis. The instrument was software-controlled, allowing assay protocols to be easily modified or updated without modification of the chip cartridge. To test the system, the authors performed both, immunoassays and DNA assays, using the same chip design on this portable platform. A magnetic bead-based immunoassay for cardio troponin I (cTn1) was developed and spiked standards were measured in both a buffer and a blood matrix. Spike recovery was evaluated and it could be noted that the immunochemical behavior on chip was not significantly different for either matrix. For further validation, the sample preparation for human genomic DNA was also performed on-chip by means of magnetic particles. Although authors successfully performed DNA extraction from blood on-chip and they had also fully demonstrated PCR on-chip, they were still underway to completely integrate both DNA extraction and PCR on the same platform. In the same context, Pollack and coworkers in 2010 presented an automated and self-contained DMF platform for multiplexed real-time PCR assays [37]. They designed an instrument of the size of a shoe-box that can be connected to a PC through a USB cable (Fig. 11B). Disposable DMF cartridges were introduced in this device, which included all the required control features such as power supply, electronics, heaters and magnets and a detection fluorimeter module. As described in Section 3, authors performed PCR amplification on this platform with remarkable efficiency, sensitivity and reproducibility, starting from spiked blood samples.




Review FIG. 11

(A) Picture of hand held instrument along with its cartridge (3.04 cm 6.09 cm) designed by Pamula and coworkers. Reprinted with permission from Ref. [53]. Copyright The Royal Society of Chemistry 2010. (B) Self-contained PCR system and disposable cartridge developed by Pollack and coworkers. Reprinted with permission from Ref. [37]. Copyright The Royal Society of Chemistry 2008 (C) Integrated platform for digital microfluidic particle-based immunoassays developed by Wheeler and coworkers. The cross-sectional images show the default and engaged positions of both PMT detector and magnet. Reprinted with permission from Ref. [57]. Copyright American Chemical Society 2013.

It proved flexibility in enabling different thermocycling conditions and protocols. However, like in the previous case, further integration with other pre-PCR or post-PCR processes is still missing. The group of Wheeler also integrated all necessary instrumentation in a benchtop device (18 23 30 cm), [57,74] (Fig. 11C). This device included a pogo pin interface, where disposable DMF chips are connected, a magnetic lens and a photo multiplier detector (PMT). It also comprised all necessary software and electronics to control droplet operation, motors and optical limit switches to manage the position of the magnetic lens and the detector. The magnetic lens consisted of a motor-driven magnet assembly comprising a neodymium magnet flanked by two steel armatures. An integrated light-emiting diode (LED) and a webcam were used to monitor on-chip droplet movement. With this integrated and fully automated platform eight magnetic separations can be performed in parallel, allowing for multiplexing. However, although manual intervention is minimal, it is still necessary to fill the reservoir electrodes with microliter volumes and, even though both immunoassays and protein depletion were successfully implemented on this platform as described in Section 16

4, analysis of real samples was never performed by the authors. It is important to emphasize that Wheeler’s lab is the first one to make their technology available in open source. As it can be seen, the trend in the field towards a greater integration is evident. However, in most of the reported examples, in which fluorescence or chemiluminescence signals are typically measured (Table 1), detection is performed off-line by either placing the DMF chip on a fluorescence microscope or by external PMT tubes coupled to the DMF chip. There are only two examples that explored alternative detection principles, but still scalable and compatible with DMF. The first one, developed by Shamsi et al. [56] consisted of integrating electrochemical detection on-chip. The electroanalytical detector, involving a gold working electrode (WE) and a silver counter/pseudoreference (CE/RE) electrode, was patterned into the device top plate by means of photolithography. In this way the top plate did not serve only as a counter-electrode for DMF droplet actuation, but also as a host to the electrochemical electrodes. They demonstrated this approach as an efficient, costeffective and simple way of integrating detection on chip. As a proof of concept they performed the previously developed TSH



immunoassay. Although the sensitivity was lower relative to the one obtained with chemiluminiscent detection [55] (2.4 mIU/ml vs. 0.15 mIU/mL), the limit of detection is sufficiently lower than the clinical cut-off value in thyroid disease diagnostics. The second alternative detection example is the Halls sensor integrated by Bhalla et al. As explained in Section 4, in their platform magnetic particles were employed as labels, indicating the presence of the analyte. The Hall sensor was integrated in the top plate of the chip. Its output voltage varied corresponding to the magnet field generated by the particles, and was proportional to the analyte concentration. These types of sensors are commercially available, inexpensive and can be easily integrated.

Commercialization Although most of the research in the DMF domain is being pursued within academia, some of the above-mentioned advanced platforms have been developed inside industrial organizations and government laboratories. Compared to academic research, commercially-driven research is limited by market demands and boundaries. Therefore, only few companies have commercialized DMF-based research and diagnostic products. An example is Advanced Liquid Logic a company founded in 2004 by Pamula and Pollack, authors of some of the most relevant publications reviewed in this paper [37,52,53]. This company launched in 2011 its first product in collaboration with NuGEN Technologies, which consisted of a workstation using disposable DMF cartridges to automate genomic-sample preparation for DNA NGS. The company is continuously growing and was recently acquired by Illumina Inc. Another successful story is the one of Sandia National Laboratories, established within the Department of Energy of the Unites States. These Laboratories have developed an Automated Molecular Biology (AMB) system which enables the cost-effective automation of complex protocols in a portable format. The AMB system is based on DMF platform which functions as a central hub for the distribution and manipulation of samples and reagents and until date its main application is also the preparation of genomic DNA for NGS [46,47] (see Section 3). Its estimated price is 2500 euros, however the Laboratories are focused on licencing their technology to potential partners rather than selling it. Many other types of microfluidic-based platforms, apart from DMF, are under development. A list of companies working on microfluidics-based POC tests was compiled by Chin et al. [4].

Conclusions and outlook Since its introduction more than a decade ago, DMF has emerged rapidly as a new microfluidic paradigm and has been used for many different applications in the fields of biochemical, analytical and medical diagnostics. In this review we presented and discussed the latest developments of bio-assay implementation on DMF platforms focused on the utilization of magnetic particles, with the aim to identify and analyze the trends in the field. As it was shown in this review, DMF’s capability of handling magnetic particles plays an important role in the development of present DMF applications. Since magnetic particles can be easily manipulated in a fast and automated way, DMF provides many assets for magnetic particle-based bio-assays for which automation and miniaturization is challenging on conventional platforms. The

REVIEW

role of magnetic particles in the presented DMF platforms is similar to the one they have in classical laboratory assays. They are mainly used to capture or immobilize targets of interest such as DNA, RNA, specific antigens or antibodies and whole cells. They also provide a convenient way for concentrating these targets and transferring them between different liquid media. Additionally, in the magnetically actuated platforms, since the droplets are translated and manipulated by means of magnetic forces, the magnetic particles have the dual role of substrates for target molecule capture and actuation vehicles. Despite the number of different droplet actuation mechanisms available, EWOD-based platforms are by far the most used in the presented applications. Some interesting applications of magnetic actuated systems were presented while other actuation mechanisms remain on the level of proof-of-concept academic research. As already explained, EWOD is popular due to its high flexibility and programmability. Also, the application of superparamagnetic particles strongly prevails over the application of ferromagnetic ones. This is because in most of the reviewed work magnetic particles are used for target molecule carrier rather than actuation mediators, and therefore fast resuspension from particle’s agglomerates is more important than the exertion of a strong force on them. Regarding magnetic particle manipulation, the simplicity and cost-effectiveness of permanent magnets has strongly triumphed over more complex and expensive magnetic coil fabrication and integration on the DMF platform. Also, there is a dominance of fluorescence and chemiluminiscence detection over other analytical techniques, and efforts have been made to integrate both detection principles into miniaturized and compact platforms. Despite the indisputable success of the presented works in demonstrating different magnetic particle-based bio-applications on DMF platforms, the DMF field in general is facing several challenges that need to be overcome. First two challenges are related with the handling of ‘‘real-world’’ samples on-chip. The first one is the sample preparation step. The fact that suspended magnetic particles can be separated, washed, resuspended and mixed on a DMF device in a fully automated way potentially provides an effective sample-to-answer method. Some efforts to address this issue have been presented also in this review and some promising results have been demonstrated, however, most of the reported work so far has only addressed a part of this entire process and it is still in the proof-of-concept level. The main challenge remains in the interface of the external world with the DMF chip due to discrepancy between the volumes. The second challenge is the compatibility of real samples with effective actuation on chip. For example, in the case of EWOD-based platforms, the electrowetting effect is less effective with viscous liquids and completely ineffective at very low surface tensions, while molecular components present in physiological samples can influence both. Moreover, these components can also affect electrowetting actuation through irreversible interactions with the chip surface (biofouling). In spite of this, physiological samples such as whole blood or plasma have been shown to be compatible under specific conditions in some of the presented examples. The third challenge is linked to the limited droplet generation throughput of actual DMF devices, especially when compared to droplet-based microfluidics. Therefore in most of the described work multiplexing and parallelization are very limited. The fourth open challenge is the chip


Review



Review

fabrication cost. Most of the manufacturing methods in the reviewed papers are based on microfabrication and patterning on glass or silicon, which are relatively expensive compared to cheap mass replication technologies used for commercialized continuous flow microfluidics. Here we need to stress a great discrepancy between the simplicity and cost of EWOD-based systems and magnetic actuated DMF systems. Although, the former offer much better flexibility and have greater potential for real applications, they are also more complex and expensive. However, scientists working on the development of EWOD platforms are aware of this and the issue is already being addressed. For example both the group of Wheeler [75] and Abadian et al. [76] recently presented a cost-effective paper-based EWOD system. Admitting these limitations, DMF has already proven to be a powerful tool for miniaturization and automation in various biooriented applications. We think that DMF’s superior strength to individually manipulate single droplets in combination with magnetic particles potentially offers a unique advantage over other


existing microfluidic approaches. The field of digital assays presented in Section 6 is of particular interest. Although until today only DNA single molecule assays have been probed in DMF, the developed platform has a huge and still unrevealed potential and opens the door for detection and analysis of other targets of great interest in the diagnostics field such as proteins cellular metabolites, viruses, prokaryotic cells, cellular organelles, microvesicles, etc. In summary, we believe that the application of magnetic particles on DMF platforms will further grow and keep pushing the limits of modern biochemistry, microbiology and biomedical diagnostics.

Acknowledgements This research was financially supported by the KU Leuven Research Council (IDO-project 10/012, OT project 13/058 and Atheromix IOF-knowledge platform), Fund for Scientific Research Flanders— FWO (G.0997.11 and G.0861.14) and the European Commission’s Seventh Framework Program (FP7/2007-2013) under the grant agreement BIOMAX (project no. 264737).

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Review


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