Microfluidics for DNA analysis

2. Microfluidic DNA extraction and purification

Nucleic acid assays obviously required to use purified nucleic acids from biological cells.

DNA extraction basically requires two steps:

• Cell lysis, i.e. disrupting cell membranes and nucleus membranes to liberate their content.
• DNA purification from the unwanted sample like membrane lipids, proteins and RNA and eventually the different reagents used during the previous step.

Chemical lysis is the most common laboratory bench method because of its ease of use with no specific equipment.

This method can be used for on-chip lysis but reagents are rather stocked off-chip and then injected in the device with external flow systems (e.g. pressure controller) to perform an on-chip lysis (1,2), which complicates the making of all-integrated lysing systems.

From Privorotskaya et al., Lab on Chip, 2010 (6)

Thermal lysis is a convenient method for PCR lab-on-chip devices since the required thermal system for lysis can also be used for the following PCR steps (3).

Indeed, several systems integrating these thermal lysis and PCR steps on chips have been previously developed (4,5). Thermal lysis simply consists in exposing cells to high temperatures. Most of the time, thermoelectric materials are integrated on the chip.

Ultrasonic lysis in microfluidic devices presents the advantage to avoid local heating in pulse mode and thus maintain protein structure while enabling a simple integration (unlike chemical lysis).

Ultrasonic lysis uses ultrasound to oscillate cavitation within cells until they disintegrate. Microfluidic devices use mainly integrated piezoelectric transducers to generate this ultrasound (7,8).

 From Medimoon website (12)

Electrical lysis use high electrical fields that can disrupt the cell membrane and thus induce their lysis. Thanks to the easy integration of electrodes, electrical lysis is a common technique used in microfluidics devices (9,10).

Nevertheless, this method presents some drawbacks like the possible water electrolysis at high voltage and thus the bubble formation.

Interestingly, electrodes can also be used for cell dielectrophoresis for upstream cell sorting (11) (see our other review about label-free cell sorting).

Mechanical lysis can be carried out in many forms in microfluidic devices. It is possible to integrate sharp micro-nano objects within the microchannels to damage the cell membranes and the lyse cells (13). Pressure forces can be used as well by using for example highly pressurized quake membranes compressing cells until their lysis (14). Magnetic beads can be used as well to lyse cells by making them beat with a rotating external magnet (15). It can be observed here that mechanical lysis requires quite specific setups which may complicate their integration in more complete devices like lab-on-chip systems.

 From Quora website (16)

Most of microfluidic DNA purification techniques have adapted the macroscopic method made in extraction columns and using the DNA adsorption on silica beads under certain buffer conditions (highly ionic and with an adapted pH). After this DNA adsorption, DNA is released from silica particles with an elution buffer (low ionic buffer as water for example).

Within microfluidic devices, silica beads can thus either be blocked by mechanical obstacles (17) or silica-coated magnetic beads  (18) to easily block these particles with a magnet for the washing and elution phases.

Other types of functionalization can be used for the magnetic beads (e.g. nucleotides (19)).

Isotachophoresis used with pressure-driven flow in capillaries can also be used to efficiently isolate DNA (20).

Briefly, isotachophoresis uses the different ionic mobility between the DNA, a “slow” and a “fast” electrolyte.

Electrolytes must be chosen so that DNA has an intermediate DNA mobility and will thus focus at the interface when an electric field is applied.

A pressure driven counter-flow can be used to control the position of the focusing interface. Thus, DNA can easily be extracted from crude samples (21).

From Standford Microfluidics Laboratory Website (22)

The importance of PCR in genomic analysis leads to the development of numerous microfluidic devices for PCR. Indeed the miniaturized volume and the high surface to volume ratio enable rapid thermal transfer for rapid and integrated PCR (23,24).

It should be mentioned that PCR also requires a subsequent analysis mainly carried out by electrophoresis to check that amplicons size corresponds to the expected PCR products and thus many works have been made to integrate PCR and electrophoresis on-chip. (25,26). Microfluidic electrophoresis is discussed in the following paragraph.

Eventually, integrated microarrays can be used as well since they allow to check specifically PCR products and not just their molecular weight (27).

Interestingly, DNA processes like DNA extraction and post-PCR analysis carried out on-chip can also be combined to design total analysis systems and lab-on-a-chip devices (3,25,28).

PCR & Electrophoresis microfluidic system, From Kaigala et al., Analyst, 2010 (26)

Melting curve analysis for qPCR with intercalating agent

These fluorescent reporters can be either fluorescent intercalating agents like Sybr Green or specific fluorescent probes.

Intercalating agents react with double-stranded DNA which makes this fluorescent DNA detection non-specific but cheaper. Therefore, this type of detection requires another step to verify the amplification specificity: a melting curve analysis. During this step, fluorescence is monitored while the temperature is increased. As the temperature is raised, DNA strands dissociate and thus the fluorescence, specific to double-stranded DNA, decreases. If the derivative of the fluorescence in function of temperature reveals a single-peak, the PCR reaction can be considered as specific. If several peaks appear, it means that non-specific PCR products have been unintentionally amplified (like primer dimers for example).

Compared to PCR, qPCR presents the advantage to be faster (automated detection during PCR), more sensitive, have a much broader dynamic range and have a better repeatability.

Since the development of qPCR and the first PCR microfluidic devices, many qPCR microfluidic devices integrating optical detection are therefore rather developed (29–31).

As an example, using Elveflow ultra-fast pressure controller and fluorescence reader and based on the Elveflow ultra-fast temperature control, ELVESYS developed an ultra-fast qPCR microfluidic system for the molecular detection of diseases like Anthrax and Ebola in less than 8 minutes with a detection efficiency identical to commercial systems that are 7 to 15 times slower (32).

This system uses the improved thermal conduction of liquids compared to air, enables to reach high thermalization speeds. The device also uses the advantage of a large surface to volume ratio inherent to microfluidics to enhance thermal exchanges. In addition to ultra-fast and sensible qPCR, the system uses monophasic samples and is compatible with a broad sample volume ranging from nanoliters to tens of microliter.

Elvesys system for ultra-fast microfluidic qPCR

From Beer et al., Analytical Chemistry, 2007 (33)

With the development of microfluidics, emerged the field of digital microfluidics dealing with emulsion and droplets within microfluidic devices.

The precursor paper of Beer et al. (33) showed the interest of this technique to handle ultra-low amount of DNA within droplets and thus increase even more the detection limits obtained with qPCR. Indeed, with 10 pL droplets, a single DNA copy could be amplified. At the end of qPCR reaction, fluorescent droplets are counted and Poisson distribution is finally used to thus determine the absolute number of templates in the droplets.

Like PCR in a lower extent, DNA electrophoretic separation has many applications.

One of the main applications is the verification of PCR results like mentioned in the previous paragraph. DNA must be stained with an intercalating agent like ethidium bromide or Sybr Green and can then be seen under UV light. With the electrophoretic separation, DNA is separated by size and compared to a calibrated molecular weight ladder to check the obtained molecular weight and compare it with the expected PCR result. Even if molecular weight is verified, the molecular sequences are not, which makes this type of detection less specific than qPCR with fluorescent probes.

Electrophoresis was originally carried out in gels, mainly made in agarose (for longer DNA) and polyacrylamide (for shorter DNA). These gels are porous which explains why they decrease DNA electrophoretic mobility according to DNA size/molecular weight.

Then electrophoresis was adapted to capillaries which present several advantages over gels. First, they allow to use much higher voltage and thus reduce the time of analysis (34). Secondly, thanks to their large surface to volume ratio, they dissipate effectively Joule heat which is important not to create a temperature gradient and thus convection effect that could disturb electrophoretic effect. Also electrophoretic mobility depends on the temperature and thus a temperature gradient could cause spatial dependence of the mobility and deformation of the migrating zone (34,35).

With the advent of microfluidics, DNA electrophoresis was one the first molecular process to be integrated on a chip (36). This miniaturization enabled to increase even more the process time to realize electrophoresis (e.g. sequencing of hundreds of bases in a few minutes (37), reduce reagent consumption and assemble on a chip other DNA process steps as mentioned before. Now, microdevices for DNA electrophoresis analysis have been commercialized.

It is interesting to note that pressure control can also be used in conjunction with electrokinetic force to inject DNA sample within the separation channel (38). This technique enables to avoid the injection disparities between species with different electronic mobilities (39).

Examples of sample injection methods in electrophoresis microchips. (a) floating injection, (b) double-T mode. Adapted from Dorfman et al., Chemical Review, 2012 (34). Explanations about these injection methods can be found in this excellent review.

Microfluidics has been widely used to improve Sanger sequencing, especially because of the work made on the improvement of electrophoresis (37) and the integration of other sequencing steps (DNA purification and PCR) (cf previous paragraphs) (40).

Next generation sequencing technologies correspond to techniques that enable high-throughput sequencing to reduce drastically its cost and duration. The developed systems use globally micro-nanotechnologies, biophysical and biochemistry techniques rather than specifically microfluidics even if milli and microfluidics are intrinsically required to easily transport and exchange reagents within consumables.

Among the most famous next-gen sequencing systems, we can name Illumina and its sequencing by synthesis technhology, Life technologies and its Ion Torrent using semiconductor technologies, Pacific Bio with its single molecule real time sequencing using zero-mode waveguides, Roche with its 454 sequencing system using pyrosequencing technology.

Nevertheless, microfluidics is more straightforwardly used in other systems that rely on the sequencing technic initially based for microarray (41), i.e. hybridization sequencing. Basically, a sequence to be identified is exposed to large set of short labeled probes (typically 5 to 10 nucleotides) and their hybridization is thus optically detected after having washed away the non-hybridized probes. Either the studied sequence or the probes can be fixed on the microarray.

The same principle has been applied by placing the different labeled probes within droplets and exposing them to a DNA sequence. The hybridization is then optically detected by scanning each droplet (42).

Microfluidic devices have been developed to automatically prepare the sequencing library (43), i.e. the randomly fragmented DNA sequences that will be then used for the following sequencing steps. Devices for automated library preparation are now also commercialized by sequencing companies.

Microfluidic Lab on chip device for Sanger sequencing gathering thermal cycling, sample purification and electrophoresis on chip. From Blazej et al., PNAS, 2006 (40)