Rapid immunodiagnostics of multiple viral infections in an acoustic microstreaming device with serum and saliva samples

Abstract

There is a growing need to screen multiple infections simultaneously rather than diagnosis of one pathogen at a time in order to improve the quality of healthcare while saving initial screening time and reduce costs. This is the first demonstration of a five-step protein array assay for the multiplexed detection of HIV, HPV and HSV antibodies on an integrated microfluidic system. HIV, HPV and HSV reactive antibodies from both serum and saliva were rapidly detected by acoustic streaming-based mixing and pumping to enable an integrated, rapid and simple-to-use multiplexed assay device. We validated this device with 37 serum and saliva samples to verify reactivity of patient antibodies with HIV, HPV and HSV antigens. Our technology can be adapted with different protein microarrays to detect a variety of other infections, thus demonstrating a powerful platform to detect multiple putative protein biomarkers for rapid detection of infectious diseases. This integrated microfluidic protein array platform is the basis of a potent strategy to delay progression of primary infection, reduce the risk of co-infections and prevent onward transmission of infections by point-of-care detection of multiple pathogens in both serum and oral fluids.

Figure 1:. Schematic and layoutof an integrated microfluidic device for multiplexed detection of viral infection.

a) Device schematic showing LCAT’s and VCAT’s for pumping and mixing of patient sample containing antibodies aligned on the nitrocellulose pad. b) PDMS device bonded on top of the nitrocellulose pad, ready for use. c) Layout of HSV, HIV, HPV antigens produced by IVTT and HIV antigens produced in eukaryotic cells as well as primary and secondary IgG and IgA and negative controls. d) Plot of normalized spot intensity for reactivity against various spots at different sample incubation times. e) Plot of normalized intensity for reactivity against HSV+ spots for devices with and without VCAT mixers.

Figure 2:. Serum antibody reactivity with HSV, HIV and HPV proteins on microarray.

a) Microarrays following 17.5minute assay with (i) triple negative donor sera (n=4 patient samples, two repeats), (ii) HSV+ donor sera (n=4 patient samples, two repeats), (iii) HIV+ donor sera (n=5 patient samples, two repeats), and (iv) HIV+, HPV+ sera (n=5 patient samples). b) Heat map of serum antibody reactivity with the color histogram on right indicating intensity on a log base 2 scale.

Figure 3:. Most significantly reactive proteins with positive serum antibodies in comparison to triple negative serum samples:

a) Reactivity of HSV+ serum samples with HSV proteins on the microarray. b) (i) Reactivity of HIV+ serum samples with HIV proteins produced by IVTT, (ii) Reactivity of HIV+ serum samples with HIV+ proteins produced in eukaryotic cells, c) Reactivity of HIV and HPV double positive serum samples with HPV IVTT proteins. *** denotes significance with p−4, ** indicates significance for p<0.05, * shows significance for p<0.1. Y-axis displays the average intensity of the spots with log base 2.

Figure 4:. Saliva antibody reactivity with HSV, HIV and HPV proteins on microarray.

a) Microarrays following 17.5 minute assay with (i) triple negative donor saliva (n=4 patient samples), (ii) HSV+ donor saliva (n=6 patient samples), (iii) HIV+ donor saliva (n=4 patient samples, two repeats), and (iv) HIV+, HPV+ saliva (n=5 patient samples). b) Heat map of saliva antibody reactivity with the color histogram on right indicating intensity on a log base 2 scale

Figure 5:. Most significantly reactive proteins with positive sample saliva antibodies in comparison to triple negative serum samples.

a) Reactivity of HSV+ saliva samples with HSV proteins on the microarray b) (i) Reactivity of HIV+ saliva samples with HIV proteins produced by IVTT, (ii) Reactivity of HIV+ saliva samples with HIV+ proteins produced in eukaryotic cells, c) Reactivity of HIV and HPV double positive saliva samples with HPV IVTT proteins. *** denotes significance with p−4, ** indicates significance for p<0.05, * shows significance for p<0.1. Y-axis displays the average intensity of the spots with log base 2.