The last 20 years have witnessed the explosion of technology miniaturization. Advancements in materials science, engineering, and microfabrication have led the transformation from macroscale, to microscale, and ultimately to nanoscale technologies. Micro and nanoscale technology innovations have fueled new applications across the spectrum of biomedical and molecular medicine. Scaling down from the macro to the microscale level has major advantages for analyzing events, such as discrete biomolecular processes and interactions. However, reducing the size of materials and the volumes of solutions involved in these investigations brings challenges.
The structures and chemistry of the contacting materials influence the behavior of molecules in solution. Flow rate, pressure, temperature, and evaporation impact the integrity of solutions, which can challenge the analysis, and ultimately the quality, of the data. These considerations shed light on an interesting and important point regarding the challenges of biomolecules in solutions—how does one ensure sample integrity is maintained when the scale of the experiment is significantly reduced? To address this question, we can gain insight from two state-of-the-art microscale techniques and their important biomedical research applications.
Macroscale: Anything greater than ~100 micrometers in size, and visible to the naked eye
Microscale: Ranges from 100 micrometers to 100 nanometers
Nanoscale: Ranges from 100 nanometers to 1 nanometer, or the scale of atomic resolution
Microvolume: In this context, ranges from 100 microliters to 100 nanoliters
Nanovolume: Ranges from 100 nanoliters to 1 nanoliter
Microfluidics and digital polymerase chain reaction (PCR)
PCR technologies have successfully addressed many challenges in research and medicine. Several new screening diagnostics use PCR-based liquid biopsy as a platform for detecting genetic patterns indicative of cancer. Genetic disorders that entail rare DNA variants or single nucleotide polymorphisms (SNPs) also harness the power of PCR-based diagnostics. The sensitivity and accuracy of PCR are key attributes for genetics-based diagnostics. Traditional PCR methods can struggle with complex sample backgrounds, amplification bias, and the demands of quantitation.
Digital PCR (dPCR) arose from the observations that PCR could be accomplished using a single copy of DNA. dPCR measures discrete PCR amplification reactions using single molecules of DNA, whereas conventional PCR measures the sum of many amplifications at a time in one reaction. Quantitation of dPCR is absolute, while conventional PCR relies on relative comparison with internal standards and use of a standard curve. PCR efficiency bias, arising from preferential amplification of high-copy DNA in a sample, is eliminated in dPCR. The impact of background interferences is minimized due to the sample dilution process during dPCR analysis.
A requirement for dPCR is separation of a sample into discrete reactions of single DNA molecules, and the incorporation of microfluidics and microscale partitioning technologies is critical. With partitioning into thousands of nanovolume droplets, digital measurements are taken and individual DNA molecules identified through yes or no results. Rare DNA sequence detection, single-cell analysis, and next-generation sequencing are all enabled by microfluidics and nanovolume partitioning in dPCR.
“Micro and nanoscale technology innovations have fueled new applications across the spectrum of biomedical and molecular medicine.”
Partitioning and handling of DNA samples are required for successful analyses. Microfluidic channels and partitioning within the dPCR instrument into tens of thousands of individual reactions ensures that the sample background is sufficiently dilute, and free from interfering materials and other activities that challenge sample integrity. During detection, microfluidic channels allow individual partitioned samples to be analyzed and digital results to be collected and interpreted. This massive partitioning and detection are coupled with Poisson distribution statistics calculations to measure the absolute quantity of sample DNA.
dPCR platforms use a range of technologies to partition microvolume samples during analysis. One example involves creating a mixed sample oil emulsion during microfluidic separations. Droplets of this emulsion are deposited into nanovolume plate wells. The oil encapsulates the aqueous sample, preventing loss due to non-specific absorption or evaporation as the droplet makes its way through PCR and measurement analysis. Other technologies use micro-fabricated compartments placed within the microfluidic device trapping nanovolume samples. The compartment walls are made of non-binding materials to ensure minimal DNA loss through absorption. Other technologies involve DNA-specific magnetic beads, in which a single molecule of DNA bound to a bead is encapsulated in an oil emulsion and subjected to PCR reaction.
Regardless of the method of partitioning, the goal of these dPCR technologies is the same—to stabilize and maintain the integrity of microscale samples during analysis.
Microscale liquid chromatography
As a separation science, liquid chromatography (LC) is tasked with two competing metrics—sensitivity versus speed. LC can perform normal flow, microflow, or nanoflow separations. Normal flow is faster but less sensitive; nanoflow is slower but can be much more sensitive; and microflow falls somewhere in between. Using these techniques, tiny samples can be analyzed to reveal low abundance analytes. The creation of <2 µm bead sizes and the advent of ultra-high performance liquid chromatography (UHPLC or UPLC) technologies means that systems can handle the elevated pressures needed for higher resolution separations over shorter time scales.
However, LC separations face several challenges. First, the higher pressures involved in the technique place additional demands on the flow path of the sample. The tubing connections and junctions need to operate flawlessly at these pressures. The tubing and pump materials can impact the consistency and integrity of samples. Second, despite higher resolution separations, some analytes within the sample may fail to be detected due to modification or loss during flow through the LC system. These challenges can be particularly important for low abundant analytes and species that react with materials along the flow path of the system.
Innovative technologies have brought solutions to these problems. As sample volumes are reduced, the effective surface area in contact with the flow path is increased. Advancements in materials and fabrication methods minimize negative interactions. Systems that incorporate novel column ferrules and plumbing junctions using composite materials protect sample integrity. Other approaches use fine control of valves to minimize sample stress due to pressure differentials. Use of non-reactive and non-binding surface materials or coatings throughout the system addresses sample loss or reactivity. A good example is solving the challenges of metal-binding compound resolution. Phosphate and carboxylate containing compounds interact with metal surfaces, resulting in analyte depletion or adduct formation during sample runs. Oligonucleotides, acidic peptides, cellular metabolites, and other biologically relevant species all exhibit this behavior. These interactions result in loss of low abundance species or reaction and adduct formation which subsequently escapes detection by LC or LC-MS.
Regardless of the method, coating, or material, the goal of these chromatographic technologies is the same—to stabilize and maintain the integrity of microscale samples during analysis.
dPCR and microscale chromatography exemplify innovative approaches toward minimizing the negative aspects of reducing the scale of these analyses. Both technologies strive to maximize the integrity of analytes in solution by minimizing effects of surface binding interactions, pressure and flow changes, evaporation, and temperature fluctuations during analysis. Microscale and nanoscale technologies are growing in abundance and application in the biomedical world. As such, innovations in microscale research, diagnostics, therapeutics, and other fields must continue to address and conquer the challenges of microscale samples in solution.