Many conventional sample-prep approaches are based on the idea of removing unwanted bulk from a sample, usually in progressive steps, until nothing remains except the analyte of interest. Many newer sample-prep methods are improvements on this strategy. However, advances in microfluidics technology have opened up new options in direct testing from crude samples.
Genefluidics has developed a direct-detection platform for biomolecules that it reported has sensitivity in the sub-femtomolar range for DNA and RNA and sub-picogram range for protein. The company’s electrochemical sensor cartridge is designed to work with Asklepios’ point-of-care system. The sample matrix can be blood, saliva, urine, or a tissue sample, but matrix effects are minimized because of the unique nature of the capture technology.
All of the sample-prep steps that would normally be done at the bench happen inside the cartridge, which is modular with configurable components depending on assay requirements. The sensor is based on a self-assembling monolayer, which blocks nonspecific binding and provides a smooth anchor surface for probe attachment. Once the enzyme is immobilized onto the surface via the captured target, the enzymatic redox reaction generates 20,000 electrons per second from each enzyme molecule. This allows detection of a small number of target molecules.
The electrokinetic properties of the device can be customized for the sample. “We can optimize the AC frequency or amplitude to attract certain cells based on the dielectric property of the cells and also the size. It also depends on the connectivity of the specimen,” said Vincent Gau, Ph.D., CEO and president.
For example, when processing urine samples, the impedance of the sample will vary depending on whether the patient has had a lot of water to drink. Likewise, varying levels of protein in the blood will affect the impedance of the sample. Each assay begins with an impedance-matching step to select the correct current for the sample. “We identify the electrokinetic conditions to do the specific test we want to do,” Dr. Gau added.
Liviu Movileanu, Ph.D., assistant professor in the structural biology, biochemistry, and biophysics program at Syracuse University, talked about his group’s work with nanopores at the meeting. Using a combination of techniques from nanotechnology, biomolecular engineering, and surface chemistry his team is developing a chip platform for analysis of biomolecules. The sample-prep approaches previously discussed all depend on miniaturizing, automating, or simplifying the process. Dr. Movileanu said that sample prep can be effectively bypassed using an extremely sensitive detection method based on nanopores.
Natural ion channels that transport charged molecules through a potentiated membrane inspired the concept behind Dr. Movileanu’s research. Increasing understanding of the mechanisms of ion channels, and the ability to measure the current running through them, have enabled scientists to replicate them in the laboratory by creating nanometer-scale holes in a silicon nitride membrane.
When placed in an electrolyte solution with voltage across the membrane, these holes behave similarly to a natural ion channel. More importantly, the current measured when an analyte passes through a nanopore positively identifies that analyte—a technique called stochastic sensing.
“This is a technique for probing very minute, small quantities of biologic material, in this case, proteins or nucleic acids. It’s called stochastic sensing because each molecule interacting with a single nanpore will cause a current blockade. The nature of that current blockade is stochastic. The technique allows quantification, as well as identification of the analyte,” said Dr. Movileanu.
Applications for stochastic sensing include DNA sequencing and protein detection. For example, it’s possible to modify the nanopores for studying aptamers. Then, when the proteins bind to the aptamers, they create a current blockade that detects the presence of the proteins.
Microscale fabrication plays an important role in this year’s crop of sample-prep innovations. Smooth surfaces, high-tech materials, and precision design make it possible to fit an entire workflow on a chip, cartridge, or handheld device. Innovative approaches to filtration and concentration can bridge the gap between the real world and the microworld on the chip.
Although the end-user applications can be extremely different, sample-prep technologies overlap significantly between environmental, biodefense, and medical research fields. For this reason, biological researchers have benefitted tremendously by investment in sample-prep technologies for biodefense applications.