Thrust 2: Technologies for sample preparation

Sample pre-processing is a crucial step in pathogen diagnostic assays, which impacts the accuracy and sensitivity of subsequent detection. Sample processing faces three major challenges. First, raw bio-samples contain not only pathogens but also interfering materials such as blood cells, debris, nonspecific proteins (fibrinogen, albumin), and nonpathogenic nucleic acid sequences. It is critical to isolate the target from the interferants because interferants can detrimentally affect the analysis, causing false negative and false positive errors. Second, the target concentration (such as virus, antigen, or pathogenic DNA) in raw samples may be extremely low, and can extend down to a single copy in a small volume test sample, necessitating pre-concentration of large sample volumes or molecular amplification. Third, the predominant lysing methods (such as chemical and thermal) can damage target molecules (such as viral spike proteins, RNA, or DNA) causing detection approaches that require precise molecule recognition to be ineffective.

High aspect ratio and hierarchical nanostructures for pathogen lysis

Traditional nanofabrication derives from semiconductor manufacturing, feature thin films and low aspect ratio structures. Among the methods for high aspect ratio nanostructure fabrication, deep reaction silicon etching (DRIE) requires expensive equipment while limited to features larger than 500nm due to sidewall scallops. Metal-assisted chemical etching (MacEtch) was pioneered by Prof. Xiuling Li as an alternative to DRIE. MacEtch structures provide high aspect ratio (>100:1), and low cost, without requiring expensive semiconductor manufacturing equipment. In this aim, we will explore two strategies: The first strategy increases the Height/Diameter (H/W) ratio, while the second approach increases surface area by adding nanostructures to the sidewalls of micropillars by the ratio of H1/W1. Both approaches increase the surface area facilitating the capture or isolation of molecules or intact pathogens.

We plan to design and fabricate MacEtched structures for viral isolation, through high aspect ratio structures that can effectively capture objects in the 50-120 nm diameter range. MacEtch combined with nanopatterning technology such as electron beam lithography or nanoimprint lithography can significantly increase the throughput of viral isolation from serum to more than 1mL/hour.

Reagentless mechanical lysing

Lysing is a significant sample preparation step for pathogen detection. Bacteria and viruses can be specifically identified by analyzing nucleic acid molecules inside their membrane. DNA and RNA extraction requires disrupting the membranes, followed by an assay procedure performed on the released contents. Current lysing methods use chemical agents or high temperatures to break the bacterial/viral membrane, which can change the molecular structures of the target and cause errors in subsequent analysis steps. For example, the polymerase enzyme used for the polymerase chain reaction (PCR) is inhibited by chemicals used in the chemical lysing step, resulting in false negative test results; Similarly, protein purification and analysis can be affected by detergents such as sodium dodecyl sulfate (SDS) used for chemical lysing. Reagentless mechanical lysing using membrane disruptive nanostructures can solve this problem by eliminating the need for chemicals or heating. We will explore the use of sharp nanostructures produced by MacEtch fabrication. Structures that can easily penetrate cell membranes and (through application of laminar flow) apply large shear forces will efficiently rupture bacterial membranes, and we will explore approaches that can also efficiently break viral membranes. Using computational models for viral and bacterial pathogens in contact with sharp nanostructures, we will explore the optimal lysing conditions including flow rate, nanospike geometry, and micropillar arrangement. We plan to integrate a pathogen lysing stage with sensor chips (Aim 4) to integrate multiple functions in a single microfluidic cartridge. Preliminary results from Hu’s group (ZJUI) have shown 80% efficiency of bacterial lysing and ATP extraction from E.coli bacteria24. We will explore the approach for lysing additional bacteria strains such as salmonella and listeria, as well as food-borne pathogens such as geobacillus stearothermophilus.