Centrifugal micro-fluidic biochip

The principle for the centrifugal micro-fluidic biochip includes the basic forces of a particle as well as the principle of flow control.

Pseudo-forces acting in centrifugal microfluidics. While the centrifugal force always acts radially outward, the Coriolis force acts perpendicular to both ω and the fluid velocity, and the Euler force is proportional to the angular acceleration.[12]

For a particle in the flow the basic forces are centrifugal force, Coriolis force, Euler force and viscous force.

The centrifugal force plays a role as a pump in the fluid flowing. It offers the basic source to transfer the fluid flowing from the inner radius of CD to the outer radius. The magnitude of the centrifugal force is determined by the radius of particle location and the rotational speed. The formula for centrifugal force density is:

${\displaystyle f_{\mathrm {\omega } }=N{\omega }^{2}r.}$

where N is the mass density of the liquid, ω the angular frequency and r the (radial) distance between the particle and center of the disk.

The formula for Coriolis force density is:

${\displaystyle f_{\mathrm {C} }=2N{\omega }u.}$

where u is the flow velocity.

The Coriolis force generates when the liquid has a velocity component along the radial direction. This force is generally smaller than the centrifugal force when the rotating speed is not high enough. When it comes to a high angular frequency, the Coriolis force makes a difference to the flow of liquid, which is often used to separate fluid flow in the separation unit.[13]

Another basic force is Euler force, which is often defined as the acceleration of angular frequency. For example, when the CD is rotating at a constant speed, the Euler force is relatively slow. The formula for Euler force density is:

${\displaystyle f_{\mathrm {E} }=Nr{\frac {d{\omega }}{dt}}.}$

As for a particle in the fluidic flow, the viscous force is:

${\displaystyle f_{\mathrm {v} }=v{\frac {\partial ^{2}u}{\partial x^{2}}}.}$

v is the viscosity of the liquid.

As for the entire fluid flow, surface tension plays an important role in flow control. When the flow comes across a varied cross section, the surface tension will balance the centrifugal force and as a result block the flow of liquid. Higher rotation speed is necessary if the liquid would like to enter the next chamber. In this way, due to surface tension, the flowing process is divided into several steps which makes it simpler to realize flow control.

There are various typical units in a centrifugal microfluidic structure, including valves, volume metering, mixing and flow switching. These types of units can make up structures that can be used in a variety of ways.

Passive valves solely actuated by centrifugal forces: (a) capillary, (b) hydrophobic, (c) burstable seal, (d) centrifugo-pneumatic overpressure, (e) centrifugo-pneumatic under pressure, (f) remotely vented collection chamber (e.g., by wetting a dissolvable film56), (g) remotely vented inlet chamber (e.g., by a clepsydra structure), (h) capillary siphon, (i) overflow siphon, and (j) pneumatic siphon valve.[12]

The principle of valves is the balance between centrifugal force and surface tension. When the centrifugal force is smaller than the surface tension, the liquid flow will be held in the original chamber; when the centrifugal force overbalances the surface tension due to a higher rotating speed, the liquid flow will break the valve and flow into the next chamber. This can be used to control the flow process simply by controlling the rotating speed of the disk.

The most commonly used valves include the hydrophilic valve, the hydrophobic valve, the syphon valve and sacrificial valve.

As for hydrophilic and hydrophobic valves, the generation of surface tension is almost the same. It is the sudden change of cross section of the channel that generates the surface tension. The liquid flow will be held in a hydrophilic channel when the cross section suddenly becomes large, while the flow will be held when the cross section of hydrophobic channel suddenly shrinks.

The siphon valve is simply based upon the siphon phenomenon. When the cross section of the channel is small enough, the liquid in the chamber is able to flow along the channel due to surface tension. Unlike hydrophilic or hydrophobic valves, surface tension acts as a pump in this model while centrifugal force acts as resistance instead.

The sacrificial valve is a new technique that is controlled by laser irradiation. These sacrificial valves are composed of iron oxide nanoparticles dispersed in paraffin wax. Upon excitation with a laser diode, iron oxide nanoparticles within the wax act as integrated nanoheaters, causing the wax to quickly melt at relatively low intensities of laser diode excitation. The valve operation is independent of the spin speed or the location of the valves and therefore allows for more complex biological assays integrated on the disk.[1]

Aliquoting principle.

Volume metering is a typical function of centrifugal fluidics to reach a certain amount of liquid reagent. It can be achieved by simply connecting an overflow channel to the chamber. Once the liquid is at the level of the overflow channel, the rest of the liquid will be routed into the waste chamber connected to the overflow channel.

Mixing is an important function in microfluidics, which combines various reagents for downstream analysis. As the fluid is confined in the microscale domain, mixing becomes difficult due to the low Reynolds number with laminar flow. That indicates that there is no convective mixing but diffusion, which limits the mixing process. This problem can be solved using several methods. A typical way is to rotating the disk in different directions, namely clockwise and counter clockwise rotation.

Flow switching is necessary when routing reagents into different chambers. A common method for flow switching in a centrifugal device is to utilize the Coriolis force within a Y-shaped structure. When the rotating speed is too low, the liquid flow will follow the original path; when the rotating speed is high enough, which is at almost the same level as centrifugal force, the liquid flow will be routed into another chamber.

Other functions such as sedimentation are also used in microfluidic platforms when necessary. Due to the different mass and radius between different particles, these particles can be separated by viscosity and velocity. In this way, the sedimentation of different particles can be achieved.

Before the molecules react with the reagents, they should be prepared for the reactions. The most typical is separation by centrifugal force. In the case of blood, for example, the sedimentation of blood cells from plasma can be achieved by rotating the biodisk for some time. After separation, all molecular diagnostic assays require a step of cell/viral lysis in order to release genomic and proteomic material for downstream processing. Typical lysis methods include chemical and physical method. The chemical lysis method, which is the simplest way, uses chemical detergents or enzymes to break down membranes. The physical lysis can be achieved by using bead beating system on a disk. Lysis occurs due to collisions and shearing between the beads and the cells and through friction shearing along the lysis chamber walls.

ELISA (enzyme-linked immunosorbent assays) and FIA (fluorescent immunoassays) are two methods of immunoassays. Immunoassays are standard tools used in clinical diagnostics. These tests rely on the specific detection of either the antibody or antigen, and are commonly performed by labeling the antibody/antigen of interest through various means such as fluorescent or enzymatic labels. However, washing, mixing, and incubation always take a great deal of time. When integrated in microfluid biodisks, the detection times become extremely short and such types of tests can be widely used in this area.

In ELISA method, enzymes are used to produce a detectable signal from an antibody–antigen complex. At the first step, any antigen present will bind to capture antibodies which have been coated on the channel's surface. Then, detecting antibodies added to bind to the antigen. The enzyme-linked secondary antibody follows the detecting antibodies and binds to them. Finally, when substrate is added, it will be converted by enzyme to a detectable form. Base on this principle, Sergi Morais achieved multiplexed microimmunoassays on a digital versatile disk. This multiplexed assay could achieve detection limits (IC10) of 0.06μg/L and sensitivities of (IC50) 0.54μg/L.[15]

In addition to typical ELISA assays, fluorescent immunoassays (FIA) are also introduced on a centrifugal microfluidic device. The principle of FIA is almost the same with ELISA; the most significant difference is that fluorescence labels are used instead of enzymes.

Nucleic acid sensing using gene-specific nucleic acid amplification with a fluorescence dye or a probe, nucleic acid microarrays, such as DNA microarrays, have become important tools for genetic analysis, gene expression profiling, and genetic-based diagnostics. In the gene-specific nucleic acid amplification, standard PCR or isothermal amplification, such as loop-mediated isothermal amplification (LAMP), is used to amplify the target genetic marker with the DNA-binding fluorescence dye or a sequence-specific probe is applied for signal generation.[16] The fluorescence can be detected in a modified CD/DVD drive or a disc device.[17]

In the nucleic acid microarrays, the process of probe immobilization and signal amplification can be separated into five steps. The surface of the microchannel is first irradiated with UV light in the presence of ozone to produce a hydrophilic surface with a high density of carboxylic acid groups (step 1). Then, the probe molecules (biotin, DNA, or human plasma IgG) are covalently attached to the polycarbon surface via amide coupling (step 2). Later, the target molecules are labeled with fluorescent tags and this biotin-labeled target DNA is hybridized with the probe DNA immobilized on the disk (step 3). Subsequently, gold nanoparticles are bonded with the target via streptavidin conjugate (step 4). Silver is then deposited onto the gold “seed” (step 5) to increase the particle size from a few to several hundred nanometers. The amplification of fluorescence will be detected by the detection system in the CD drive.[7]

The detection system should be completed by the signal receiving component. There are roughly three types of systems which can be used for detecting. The first is Hardware and software modification, which means the CD/DVD drive should be modified and the software should also be developed at the same time. This type will cause superfluous labor and expenses, and may not be versatile in developing countries or indigenous areas. The second type is Software modification with standard hardware, which means that the detection can be achieved by developing customer software on platforms such as C++ without making any changes to hardware. The third is Standard hardware and existing software, which means that the detection can be realized simply by using the existing equipment. Manu Pallapa described a new protocol to read and quantify biotin–streptavidin binding assays with a standard optical drive by using a current CD-data analysis software (IsoBuster) successfully.[18] The latter two types are both considerable when coming across different situations.

No matter which type of detection system one uses, the reading method is an important factor. There are mainly two reading methods, which are AAS (acquired analog signals) and ERD (error reading detection). In the AAS method, to determine multianalytes on a DVD, the analog signals acquired directly from the photodiode of a CD/DVD drive correlate well with the optical density of the reaction products. The ERD method is based on the analysis of reading errors. It can use the same digital versatile disk and a standard DVD drive without any supplementary hardware.

In the ERD method, the position and level of the resulting reading error correspond to the physical location and the intensity of the bioassay signal, respectively. The errors are then compared with a perfectly recorded CD to identify the time when one certain error was read out. There are several free CD-quality diagnostic programs, such as PlexTools Professional, Kprobe, and CD-DVD Speed, which can be used to access the error-statistic information in a CD/DVD drive and to generate a plot displaying the variation of the block error rate as function of playtime. In a typical 700-MB CD-R containing 79.7 minutes of audio data, for example, the radius that error occurs can be calculated from the following equation:[7]

${\displaystyle r={\sqrt {{t \over 79.7}(58^{2}-25^{2})+25^{2}}}}$

t is the reading time and r is the radius location.

In the AAS method, the set of servo systems (focus, tracking, sled, and spindle servos) keeps the laser beam focused on the spiral track, and allows disc rotation and laser head motion during the scanning. The amplification/detection board (DAB) is integrated into the CD/DVD drive unit and incorporates a photosensor and electronic circuitry to amplify the RF signal extracted from the photodiode transducer. The photosensor generates a trigger signal when detecting the trigger mark. Both signals are brought to the USB2.0 data acquisition board(DAQ) for digitization and quantification.[19]