Thrust 3: Sensing sensitivity and specificity

The current diagnostic methods for pathogens are generally based on detecting their nucleic acids, antibodies and antigens. Each type of analyte has both advantages and limitations. The gold standard is to measure nucleic acids using quantitative polymerase chain reaction (qPCR). Even though recent advances such as LAMP and CRISPR-Cas have shown some promising results, almost all qPCR-based tests require sample pretreatment to extract DNA or RNA from target pathogens, which can lead to incomplete extraction or partial destruction of the intended targets, resulting in false negative errors, as well as sample cross contamination, often resulting in false positive errors. In addition, these tests often require many hours to several days as the sample has to be collected and sent to a clinical laboratory for analysis where a number of labor-intensive steps are performed. In contrast, antibody or serology tests can be used to detect immune responses in the patient. However, antibodies appear much later than nucleic acids and, in the case of SARS-CoV-2, during or even after the second week of symptoms. This limitation of sensitivity has resulted in not only delayed treatment of the patients but also more chances for the un-diagnosed patients to unknowingly spread the pathogen. Antigen tests have been developed that have enabled faster screening at the early stage of disease. However, antibodies typically used in the antigen tests exhibit cross reactivities with targets from other similar pathogens.

To our knowledge, no method has been reported that is able to differentiate infectious from noninfectious pathogens directly, particularly in early stages of the infection. A whole-cell test is particularly important in terms of deciding disinfection procedure on public surfaces, because the presence
of ex vivo nucleic acids or proteins on the surface alone does not mean that an intact infectious pathogen is present. Another major challenge in the current diagnostic methods is to identify subtypes of pathogens. Recently, the accumulation of several SARS-CoV-2 variants was observed, and the study suggests that the mutations may correlate with differences in infectability and death rates. Therefore, sensors that can differentiate different subtypes of SARS-CoV-2 will help identify potential correlations, and lead to a more through understanding of the critical mechanism(s).

To overcome the limitations of the current pathogen diagnostic tests, we propose to develop novel biosensors for direct detection of pathogens with ability to differentiate infectivity and subtypes.

Pathogen-specific DNA aptamers

Our sensor will use DNA aptamers as a novel targeting molecular probe for pathogens. DNA aptamers are generated from a large DNA library of 1015 sequences via a process called Systematic Evolution of Ligands by EXponential enrichment (SELEX). DNA aptamers, as an alternative analog to antibodies, have enhanced specificity and affinity than antibodies and with additional advantages, for pathogen detection: 1) DNA aptamers are isolated in test tubes in much shorter period (2-4 weeks) than isolating antibodies from cell lines or animals. For example, aptamers for S and N proteins of SARS-CoV-2 have already been isolated, with Kd of 400 pM. With the short development time, it is possible to rapidly develop aptamers for new subtypes of viruses or new transmissive pathogens when they emerge; 2) Lu’s group has demonstrated successful use of counter selection in SELEX to generate DNA aptamers highly specific to a particular pathogen even in the presence of a large concentration of competing targets, including the same pathogen that has been rendered noninfectious by disinfection. DNA aptamers obtained by SELEX can differentiate infectious and non-infectious virus, and can distinguish a particular subtype of pathogen against other subtypes. In this way, sensors equipped with aptamers probe molecules can detect the actively infectious virus not only in early stage of the infection, but also determine when the patient is no longer contagious. 3) once isolated, DNA aptamers can be inexpensively chemically synthesized in large scale, hence, a much higher level of batch-to-batch consistency is obtained compared to antibodies; 4) DNA aptamers are stable during storage even when dried, hence they are more suitable for use as assay reagents, especially in resource limited settings; and 5) DNA aptamers can be synthesized with large variety of conjugatable moieties at defined locations, rendering a much higher level of activity when covalently attached to biosensor transducers.

Engineered nucleic acid nanostructures

Many pathogens present unique spatial patterns of antigens on their surfaces59-61. Such patterns facilitate multivalent binding to host cells for  enhanced pathogenic infectivity. Based on this naturally occurring mechanism, we propose to use designer DNA nanostructures (DDNs) to achieve multivalent binding to the unique and repeated spatial patterns of pathogen outer coat proteins and thus achieve high binding avidity and selectivity for a specific pathogen. The DDNs with programmed sequences can fold into various 2D or 3D nanoplatforms to precisely control external ligand spacing, valency, and spatial arrangements with subnanometer precision. Hence, three DDN-based strategies will be harnessed to allow multivalent binding to pathogens. In strategy I, our team has recently designed and synthesized a star-shaped DNA architecture to display 10 Dengue virus (DENV)
envelope protein-targeting aptamers in a 2D-pattern precisely mirroring the complex spatial arrangement of DENV epitopes. DENV was chosen as a representative because its epitopes present the most complex spatial pattern among all known viruses. The “DNA star” has resulted in polyvalent, spatial pattern-matching interactions, affording dramatic improvement in DENV-binding avidity and specificity to increase affinity by ~1,000-fold compared to monovalent aptamer. Our team recently demonstrated a DNA origami as platform for nucleic acid sensing. Upon binding the pathogens via the loaded aptamers, the origami structure can specifically recognize the pathogen targets via rapidly unfolding/folding the origami structure at the nanometer scale. The resulting structural alteration is easily detected by conventional instruments, such as optical spectrometers and electrochemical biosensors; or by advanced biodetection instruments with high spatial resolution, such as AFM, nanopores, and optical microscopy. The structure of DNA origami can be designed in silico and can easily expand to a large library from 1D to 3D, from simple nanowires to complicate polyhedrons. By using DNA origami as a nanoplatform, multiple DNA aptamers can be assembled into a defined 3D spatial arrangement with optimal loading positions with DNA aptamers located at viral membrane attachment points.

Photonic metamaterials for enhanced light-matter interaction

Currently available methods for point of care pathogen diagnosis are mainly based on a few approaches: PCR, LAMP, and optical biosensor sensing (i.e., surface plasmon resonance, photonic crystals, and metamaterials). Among these, photonic sensing is the only technique which can reach high sensitivity without the need for chemical amplification. However, one key technical challenge is to fabricate large area photonic nanostructures with high fidelity. In particular, photonic crystals and metamaterials can provide sensitivity in the picomolar range, but rely on sub-wavelength periodic features. 3D photonic sensors are hypothesized as a route towards dramatically enhancement of light-matter interactions between the analytes and the sensor due to the potential to perform sensing within a large volume, rather than upon a quasi-planar surface. We will combine traditional lithographic techniques with our novel self-rolling- technique to create three-dimensional nanophotonic sensors for pathogen detection. Recently, we reported the use of a multilayer quasi-3D nanophotonic sensor to identify proteins at zeptomole sensitivity. To achieve molecular specificity, we will utilize DNA aptamers (Aim 3.1) on the planar structures to specifically target pathogens.