October 1, 2013 (Vol. 33, No. 17)

Recent years have seen a proliferation in the number and variety of molecular diagnostic tests for the detection of microbes and viruses, as well as for human diseases.

The efficacy of these tests relies heavily upon sample preparation to facilitate the downstream detection of target nucleic acids, proteins, and other analytes.

While traditional laboratory purification methods have been developed in well-equipped and staffed academic and industrial laboratories, sample-prep platforms are evolving to meet applications in increasingly diverse settings. For example, the shortage in developing countries of lab services for the diagnosis of infectious diseases such as tuberculosis and dysentery is driving the development of point-of-care (POC) diagnostic tests incorporating sample-preparation steps.

Moreover, concern over the threat of biological weapons such as anthrax has fueled military investment in the development of handheld assays for rapid field detection of microbial infection, again requiring initial sample-processing steps. Accordingly, demand is increasing for predetection sample-preparation solutions that address requirements of low cost, speed of processing, portability, and ease-of-use.

Highlighting the critical importance of effective sample-prep procedures in life science research, the Knowledge Foundation’s recent “Sample Prep” conference in San Diego featured several companies involved in the development of novel sample-purification and -fractionation platforms for molecular diagnostics and other applications.

Cell Lysis

The lysis of cells within samples is a critical first step in any sample-prep procedure, encompassing diverse variables such as pH, temperature, salt concentration, and the method of cell disruption. Claremont BioSolutions is developing a number of compact mechanical cell disruption devices, with an emphasis on speed and ease of use. The company says its core technology is based on a highly efficient cell-disruption platform.

“Cells are lysed by subjecting them to high mechanical shear forces using a 30,000 rpm motor,” explained Bruce Irvine, Ph.D., CTO. “Simultaneously, nucleic acids and proteins released from the cells bind to the lysing particles.”

The company manufactures a range of compact devices that run the gamut of the sample-prep process, from initial lysis to elution of a custom purified protein.

“Our OmniLyse® kit comprises six distinct chambers in which different steps, such as washing and eluting, can be carried out in a customized sequence. One adaptation of the OmniLyse kit incorporates a pre-packed affinity column that allows for recovery of His-tagged proteins at yields of up to 95%,” he pointed out.

“Samples can be lysed in five minutes, compared to traditional lysing methods that can take up to 50 minutes, and we realize a similar time saving in washing and eluting steps. Yields from the column are higher by 2–3 fold compared to existing purification methods, with no loss of function.”

The pumping system for Claremont Biosolution’s sample-prep devices is a disposable syringe that attaches to the cartridge. “Its design buys us a lot of microfluid capacity within a relatively simple architecture,” continued Dr. Irvine.

He went on to highlight the potential of such a small system in improving healthcare in developing countries. “The emphasis we place on ease-of-use makes affordable point-of-care diagnostics possible in regions where it is most urgently needed,” said Dr. Irvine.

Claremont BioSolutions is a subcontractor on a NIH grant developing an assay for the detection of Mycobacterium tuberculosis in sputum, and is the recipient of a $3 million SBIR grant for the development of a bench-top diagnostic platform for Clostridium difficile in stool.

No Need for Training

Integrated Nano-Technologies is another company focused on the rapidly growing field-diagnostics sector, and sample-prep features prominently in the company’s core technologies.

“We have developed a sample-preparation system that is fully automated, which negates the need for training,” noted Mike Connolly, Ph.D., president and CEO. “It can really be looked at as a portable laboratory.”

The sample-prep device comprises a disposable plastic cartridge capable of automating sample sonic disruption, magnetic separation, desalting, filtration, and chromatography, with power being supplied by a separate portable battery unit. Cartridges also incorporate an archiving chamber to preserve part of the sample for alternative analytic processes. Custom sample diagnostics can also be carried out within the cartridge.

“RNA or DNA isolated from samples has been successfully used for downstream processes, including PCR amplification and sequencing,” said Dr. Connolly. He emphasized the customizable nature of the platform pointing out that the system is designed to be highly flexible. “The ability to change the order of the steps makes it readily adaptable to a variety of applications in the field,” he continued.

Like Claremont Biosolutions, Dr. Connolly places a strong emphasis on reducing production costs to maximize the potential of the platform in developing countries.

Electricity Issues

In the most inhospitable field environments, a lack of reliable access to electricity can further complicate sample preparation. CUBRC has been working with the Department of Defense to develop rapid, inexpensive approaches to sample prep. The company recently developed a platform for the isolation of protein, RNA, and DNA from samples by solid-phase extraction without the use of electricity.

“The chromatography platform is based on disposable transfer pipettes,” explained David Pawlowski, Ph.D., senior research scientist at CUBRC. “The resin or sorbent is built into a transfer pipette and held within by a high-density polyethylene frit.”

The platform is ideal for use in high schools, or for rapid development of purification schemes on a small scale. Using this system CUBRC has successfully demonstrated solid phase nucleic acid and protein extraction using silica as the sorbent and His-tagged protein purification with commercial metal affinity resins, according to Dr. Pawlowski. “Our platform can readily accommodate other purification strategies designed around antibody conjugates, aptamers, or nucleic acid oligomers,” he said.

While the detection of bacterial pathogens in biological samples has been the traditional province of immunological methods such as ELISA, the stability and resilience of antibodies limits their reliability.

A group led by Stephane Evoy, Ph.D., at the National Institute for Nanotechnology at the University of Alberta, is developing an alternative to antibody detection of microbes based on bacteriophages.

“Bacteriophages have a highly specific relationship with strain of bacteria,” explained Dr. Evoy. “E. coli has a specific phage, as does Salmonella, and there is no cross-reaction between the bacteriophages strains other than their specific host.”

The specificity and precision of this interaction derives from sequences contained in viral tail-spike proteins, or TSPs, which engage signature carbohydrate moieties on the bacterial cell wall. Recombinant peptides representing the TSP amino acid sequences responsible for mediating interactions with the pathogens can be coupled to magnetic beads.

“The smaller size of the TSP indeed provides more uniform coverage to the capturing surface,” noted Dr. Evoy, who added that the system has demonstrated considerable advantages in terms of speed of sample processing.

“Recovery of bacteria from milk or blood can take up to 12 hours,” according to Dr. Evoy, “but this can be reduced to 30 minutes using bacteriophage-based probes.”

CUBRC has developed an electricity-free chroma-tography platform conceptually based on dis-posable transfer pipettes. CUBRC says it has suc-cessfully demonstrated solid-phase nucleic acid and protein extraction using silica as the sorbent (top) and His-tagged protein purification using Talon® (Clonetech) (middle) and His-bond® (Nov-agen) (bottom) metal affinity resins. [David Pawlowski]

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