SUMMARYThe survival of airborne human coronavirus 229E (HCV/229E) was studied under different conditions of temperature (20 + 1 °C and 6 + 1 °C) and low (30 _+ 5~), medium (50 + 5~o) or high (80 +_ 5~o) relative humidities (RH). At 20 +_ 1 °C, aerosolized HCV/229E was found to survive best at 50~ RH with a half-life of 67-33 + 8-24 h while at 30~ RH the virus half-life was 26.76 +_ 6.21 h. At 50~ RH nearly 20~o infectious virus was still detectable at 6 days. High RH at 20 _+ 1 °C, on the other hand, was found to be the least favourable to the survival of aerosolized virus and under these conditions the virus half-life was only about 3 h; no virus could be detected after 24 h in aerosol. At 6 + 1 °C, in either 50~ or 30~ RH conditions, the survival of HCV/229E was significantly enhanced, with the decay pattern essentially similar to that seen at 20 + 1 °C. At low temperature and high RH (80~), however, the survival pattern was completely reversed, with the HCV/229E half-life increasing to 86.01 + 5.28 h, nearly 30 times that found at 20 + 1 °C and high RH. Although optimal survival at 6 °C still occurred at 50~o RH, the pronounced stabilizing effect of low temperature on the survival of HCV/229E at high RH indicates that the role of the environment on the survival of viruses in air may be more complex and significant than previously thought.

Proteomics aspires to elucidate the functions of all proteins. Protein microarrays provide an important step by enabling high-throughput studies of displayed proteins. However, many functional assays of proteins include untethered intermediates or products, which could frustrate the use of planar arrays at very high densities because of diffusion to neighboring features. The nucleic acid programmable protein array (NAPPA), is a robust, in situ synthesis method for producing functional proteins just-in-time, which includes steps with diffusible intermediates. We determined that diffusion of expressed proteins led to cross-binding at neighboring spots at very high densities with reduced inter-spot spacing. To address this limitation, we have developed an innovative platform using photolithographically-etched discrete silicon nanowells and used NAPPA as a test case. This arrested protein diffusion and cross-binding. We present confined high density protein expression and display, as well as functional protein-protein interactions, in 8,000 nanowell arrays. This is the highest density of individual proteins in nano-vessels demonstrated on a single slide. We further present proof of principle results on ultra-high density protein arrays capable of up to 24,000 nanowells on a single slide.

Better and more diverse biomarkers for the development of simple point-of-care tests for active tuberculosis (TB), a clinically heterogeneous disease, are urgently needed. We generated a proteomic Mycobacterium tuberculosis (Mtb) High-Density Nucleic Acid Programmable Protein Array (HD-NAPPA) that used a novel multiplexed strategy for expedited high-throughput screening for antibody responses to the Mtb proteome. We screened sera from HIV uninfected and coinfected TB patients and controls (n ‫؍‬ 120) from the US and South Africa (SA) using the multiplex HD-NAPPA for discovery, followed by deconvolution and validation through single protein HD-NAPPA with biologically independent samples (n ‫؍‬ 124). We verified the top proteins with enzyme-linked immunosorbent assays (ELISA) using the original screening and validation samples (n ‫؍‬ 244) and heretofore untested samples (n ‫؍‬ 41). We identified 8 proteins with TB biomarker value; four (Rv0054, Rv0831c, Rv2031c and Rv0222) of these were previously identified in serology studies, and four (Rv0948c, Rv2853, Rv3405c, Rv3544c) were not known to elicit antibody responses. Using ELISA data, we created classifiers that could discriminate patients' TB status according to geography (

Viral infections elicit anti-viral antibodies and have been associated with various chronic diseases. Detection of these antibodies can facilitate diagnosis, treatment of infection and understanding of the mechanisms of virus associated diseases. In this work, we assayed anti-viral antibodies using a novel high density-nucleic acid programmable protein array (HD-NAPPA) platform. Individual viral proteins were expressed in situ directly from plasmids encoding proteins in an array of microscopic reaction chambers. Quality of protein display and serum response was assured by comparing intra- and inter- array correlation within or between printing batches with average correlation coefficients of 0.91 and 0.96, respectively. HD-NAPPA showed higher signal to background (S/B) ratio compared with standard NAPPA on planar glass slides and ELISA. Antibody responses to 761 antigens from 25 different viruses were profiled among patients with juvenile idiopathic arthritis (JIA) and type 1 diabetes (T1D). Common as well as unique antibody reactivity patterns were detected between patients and healthy controls. We believe HD-viral-NAPPA will enable the study of host-pathogen interactions at unprecedented dimensions and elucidate the role of pathogen infections in disease development.

We report a device to fill an array of small chemical reaction chambers (microreactors) with reagent and then seal them using pressurized viscous liquid acting through a flexible membrane. The device enables multiple, independent chemical reactions involving free floating intermediate molecules without interference from neighboring reactions or external environments. The device is validated by protein expressed in situ directly from DNA in a microarray of ~10,000 spots with no diffusion during three hours incubation. Using the device to probe for an autoantibody cancer biomarker in blood serum sample gave five times higher signal to background ratio compared to standard protein microarray expressed on a flat microscope slide. Physical design principles to effectively fill the array of microreactors with reagent and experimental results of alternate methods for sealing the microreactors are presented.