Nanoplasmonic Quantitative Detection of Intact Viruses from Unprocessed Whole Blood

Infectious diseases such as HIV and hepatitis B pose an omnipresent threat to global health. Reliable, fast, accurate, and sensitive platforms that can be deployed at the point-of-care (POC) in multiple settings, such as airports and offices, for detection of infectious pathogens are essential for the management of epidemics and possible biological attacks. To the best of our knowledge, no viral load technology adaptable to the POC settings exists today due to critical technical and biological challenges. Here, we present for the first time a broadly applicable technology for quantitative, nanoplasmonic-based intact virus detection at clinically relevant concentrations. The sensing platform is based on unique nanoplasmonic properties of nanoparticles utilizing immobilized antibodies to selectively capture rapidly evolving viral subtypes. We demonstrate the capture, detection, and quantification of multiple HIV subtypes (A, B, C, D, E, G, and subtype panel) with high repeatability, sensitivity, and specificity down to 98 ± 39 copies/mL (<i>i.e</i>., HIV subtype D) using spiked whole blood samples and clinical discarded HIV-infected patient whole blood samples validated by the gold standard, <i>i</i>.<i>e</i>., RT-qPCR. This platform technology offers an assay time of 1 h and 10 min (1 h for capture, 10 min for detection and data analysis). The presented platform is also able to capture intact viruses at high efficiency using immuno-surface chemistry approaches directly from whole blood samples without any sample preprocessing steps such as spin-down or sorting. Evidence is presented showing the system to be accurate, repeatable, and reliable. Additionally, the presented platform technology can be broadly adapted to detect other pathogens having reasonably well-described biomarkers by adapting the surface chemistry. Thus, this broadly applicable detection platform holds great promise to be implemented at POC settings, hospitals, and primary care settings

Microchannel formation by spatially bioprinting the sacrificial living porogens using a cell bioprinter.

<p>(<b>a</b>) <i>E. coli</i> (800,000 CFU/mL) were mixed with 0.5% agarose at 40°C. Agarose-bacteria mixture was printed on a 1% agarose pre-coated petri dish and covered with another 1% agarose layer on top. (<b>b</b>) Top view of bacterial colony chain in 0.5% agarose. (<b>c</b>) Merged bacterial colonies. (<b>d</b>) Cross-section of a formed microchannel in the hydrogel. (<b>e</b>) Diffusion enhanced in bioprinted microfluidic hydrogels at areas with high porous density.</p

Biocompatibility of fabricated porous hydrogels.

<p>After lysis of porogens, the porous scaffolds were washed with DPBS and cell medium. 3T3 were then seeded on the scaffold and cultured. 3T3 cells proliferated and were confluent on day 6 for 1% hydrogel (<b>a–c</b>) and on day 14 for 2% hydrogel (<b>d–f</b>). Cells were alive after confluence (<b>g–h</b>).</p

Illustration of the fabrication steps of microporous hydrogel scaffolds using living sacrificial porogens.

<p>(<b>a</b>) <i>E. coli</i> encapsulation in hydrogels as porogens and l<b>iv</b>e sacrificial pore formation. <i>E. coli</i> cultured on LB agar plate were collected and mixed with the agarose solution. After mixing, <i>E. coli</i> suspension was poured into a 12-well plate and solidifies. <i>E. coli</i> encapsulated in hydrogels were continuously cultured to allow formation of colonies. The living porogens were then lysed and the debris of <i>E. coli</i> and its DNA were removed by sequential washing with DPBS and DI water. (<b>b</b>) Formation of microfluidic channels. A line of <i>E. coli</i> / agarose mixture solution was printed onto Petri dish pre-coated with a layer of agarose. Then, another layer of agarose was used to cover the bacterial line. The hydrogels were gelled under rapid cooling (4°C) overnight.</p

The mechanical stiffness (a–b) and perfusion properties (c–f) of porous hydrogels.

<p>Compressive moduli were inversely correlated with the culture time for 1% hydrogel (<b>a</b>), whereas there was no significant change in stiffness for 2% hydrogel (<b>b</b>). Fluorescence images of FITC-dextran (0.25 mM, 20 kDa) diffusion in the 1% porous agarose hydrogels (<b>c</b>) and controls (<b>d</b>). The diffusion profiles of FITC-dextran as a function of distance from the source channel in porous (<b>e</b>) and non-porous (<b>f</b>) hydrogels. (n = 3).</p

Characterization of living porogen growth in hydrogels and subsequent pore formation.

<p>Crystal violet staining of bacterial colonies in 1% (<b>a</b>) and 2% (<b>b</b>) hydrogels. SEM images of pores created using living porogens (<b>c</b>) as compared to controls (<b>d</b>). The colony size (<b>e–f</b>), density (<b>g–h</b>), and the pore area percentage (<b>i–j</b>) are presented over the culture time for 1% and 2% hydrogels at initial bacterial seeding concentrations of 9.5×10⁷, 1.9×10⁸ and 3.8×10⁸ CFUs/ml.</p