April 15, 2013 (Vol. 33, No. 8)
Horacio Kido, Ph.D.
New System Can Be Used in Field or Laboratory
One of the largest bottlenecks in a modern life science laboratory workflow is at the beginning of the sample-preparation stage: It is the efficient lysis, homogenization, and grinding of difficult-to-lyse samples. This step is necessary in order to release subcellular and nuclear content for further analysis and workflows, such as to make nucleic acids accessible for purification, amplification, sequencing, or get proteins and metabolites for further analysis.
The main requirement from the life science lysis process is that the fragile macromolecules be released in as intact a form as possible within a reasonable time-frame without introducing additional chemicals to the system, which may interfere with downstream processing. Due to the absence of additional substances such as enzymes, detergents, or corrosives, mechanical sample lysis is the preferred process for use in the preparation of difficult-to-lyse samples.
Mechanical sample lysis can be performed in a variety of ways, with the two most common techniques being the rotor and stator type homogenizers, and a variety of bead-beating systems. The disadvantage of the rotor and stator type of homogenizers is in their inability to process samples that are not already viscous or fluids, or whose outside diameters are larger than the gap between the rotor and stator, making this technique difficult for processing hard samples like most agro-biotech samples including seeds, plants, etc, as well as for many solid tissues.
In addition, the rotor and stator homogenizer cannot perform lysis in a dry state, which is the preferred method for some types of analysis. Bead-beating systems use hard mineral or metallic beads to impact a sample and break it mainly by cascade impaction. Mechanical bead beating-based homogenization can be done in a dry or wet state.
The most efficient bead-beating systems are the ones that create a strong multidirectional force field, making it possible for three different grinding processes to simultaneously act upon a sample. These processes are 1) cascade impaction, a hammer-like process of beads beating on a sample, 2) mechanical shearing by sharp edges of lysing beads travelling through the enclosed volume of a sample container and colliding with samples at different trajectories with different velocities and if lysis/homogenization is performed in a buffer, i.e., liquid media, 3) the shearing of the vortex flow generated due to the multidirectional abrupt shaking of the fluid.
There are several well-established commercial systems that perform sample homogenization, grinding, and lysis of the most difficult samples within the time-frame of 20–40 seconds based upon bead beating in an omni-directional force field; however, all of them are priced in a range from $6,000 to $20,000 in the U.S. market and all are benchtop models, not amendable for field use.
In this article we describe a novel system for ultra-fast sample lysis, homogenization, and grinding based upon omnidirectional bead beating in a handheld tool-like format, for the first time thus allowing processing of the most difficult samples, such as corn kernels, seeds, and soil in situ. This novel, patent-pending system is ultra-fast and can perform complete, quantitative dry or wet homogenization of a corn kernel in five seconds. It can be also considered as an affordable high-throughput sample-preparation lysis system for both field and laboratory use.
The MonoLyser is presented in Figure 1. It is an attachment that fits on any Dremel® rotary tool. The system is based upon multidirectional bead beating, utilizing a balanced crankshaft mechanism (patent pending). Attachment to the Dremel tool is done via a built-in threaded adapter. Dremel rotary tools are available in DC (battery)-powered versions for field use or AC-powered versions, which are ideal for continuous use in a laboratory setting. The newer versions come with an on/off switch separate from the speed control, making it easy to run multiple samples at the same speed.
A sample in a 2 mL disposable tube filled with bead-beating lysing matrix is snapped into the tube holder, and the safety door is closed. By activating the Dremel tool at 2/3 of maximum speed setting for five seconds, holding it by hand, or placing it on a laboratory stand, the complete dry or wet grinding of the even most difficult sample is quantitatively performed.
Figure 2 shows the contents of the tube after dry grinding of a dry corn kernel, one of the most difficult agro/bio samples to process. In order to demonstrate the quality of lysate, we performed complete DNA purification followed by analysis with a NanoDrop spectrophotometer and a BioAnalyzer system from Agilent Technologies. We used an MP Bio Matrix A tube with two ¼ inch ceramic balls for grinding and a Zymo Research Plant/Seed DNA MiniPrep for extraction and purification.
After lysis, the sample was centrifuged, and the supernatant was processed manually through the kit using a bind-wash-elute column based protocol described in the instructions of the kit. The resulting DNA BioAnalyser image is presented in Figure 3, together with the DNA quantification data.
At five seconds, the high quality and quantity of extracted DNA with low shearing is evident. At the longer grinding times, increasing amounts of shearing of DNA as well as decreasing average sizes were observed. This is the shortest time from hard and difficult sample to lysate in the industry. Comparable high-end benchtop homogenizers perform the same task in approximately 20–40 seconds. The quality and quantity of the extracted DNA in a five second run is comparable to those obtained by the most current lab bench high-throughput homogenizers based upon multidirectional bead beating.
We have developed a novel ultrafast high-performance high-throughput homogenizer that can break any samples without breaking the laboratory budget. The MonoLyser is the first handheld high-performance sample-preparation system that can be used equally well in the field or in a laboratory, both by holding it by hand, or by placing it on a laboratory stand. The system is capable of lysing most samples within a 2–5 seconds time frame, which makes it at least four times faster in performing homogenization of difficult samples than the next high-end system, thus allowing extractions of high-quality macromolecules without exposing them to high temperature or extended shearing time.
Furthermore the system can be utilized in a high-throughput fashion with a theoretical throughput of up to 20 samples per minute, which puts it in the range of throughput of most popular high-priced benchtop multidirectional bead beating systems, for a fraction of their list price.
Horacio Kido, Ph.D. ([email protected]), is founder and CEO of RotaPrep. Website: www.rotaprep.com. Dremel® is a trademark of Robert Bosch Tool Corporation. RotaPrep is not affiliated with Robert Bosch Tool Corporation or its subsidiaries.