Paper biosensors for detecting elevated IL-6 levels in blood and respiratory samples from COVID-19 patients

Abstract

Decentralizing COVID-19 care reduces contagions and affords a better use of hospital resources. We introduce biosensors aimed at detecting severe cases of COVID-19 in decentralized healthcare settings. They consist of a paper immunosensor interfaced with a smartphone. The immunosensors have been designed to generate intense colorimetric signals when the sample contains ultralow concentrations of IL-6, which has been proposed as a prognosis biomarker of COVID-19. This is achieved by combining a paper-based signal amplification mechanism with polymer-filled reservoirs for dispensing antibody-decorated nanoparticles and a bespoken app for color quantification. With this design we achieved a low limit of detection (LOD) of 10^-3 pg mL^-1 and semi-quantitative measurements in a wide dynamic range between 10^-3 and 10² pg mL^-1 in PBS. The assay time is under 10 min. The low LOD allowed us to dilute blood samples and detect IL-6 with an LOD of 1.3 pg mL^-1 and a dynamic range up to 10² pg mL^-1. Following this protocol, we were able to stratify COVID-19 patients according to different blood levels of IL-6. We also report on the detection of IL-6 in respiratory samples (bronchial aspirate, BAS) from COVID-19 patients. The test could be easily adapted to detect other cytokines such as TNF-α and IL-8 by changing the antibodies decorating the nanoparticles accordingly. The ability of detecting cytokines in blood and respiratory samples paves the way for monitoring local inflammation in the lungs as well as systemic inflammation levels in the body.

Keywords: Biosensor; COVID-19; IL-6; Paper-based; SARS-CoV-2; Smartphone.

Graphical abstract

Fig. 1

Photographs of the paper immunosensor and key analytical steps for detecting IL-6; (A) The biosensors consists of a piece of paper containing antibody-decorated nanoparticles, and a paper strip with three capture sites (CS); (B) After adding a drop of sample in each capture site and drying it, the paper is folded; (C) After removing the reservoir, a colored spot appears whose pixel intensity is the sum of the colorimetric signal generated in each capture site; (D) After washing away excess reagents the colorimetric signal is measured with a previously developed smartphone app within seconds; (E) Unfolding the paper strip in (C) reveals that nanoparticles are transferred vertically to the 3 binding sites; (F) Schematic representation of biomolecular interactions; IL-6 is adsorbed onto the paper substrate and subsequently detected by antibody-decorated gold nanoparticles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Colorimetric signal obtained from samples containing 0 or 10 pg mL⁻¹ when using 3 capture sites (CS) and the paper is folded so that the 3 signals add up (red), or unfolded and measuring only CS1 (black); (A) Photographs of the assays; (B) colorimetric signal for 10 (dots) and 0 (squares) pg·mL⁻¹; (C) Specific signal after subtracting 0 from 10 pg·mL⁻¹; Error bars are the standard deviation (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Detection of IL-6 in PBS (A), or spiked into blood (B), or bronchial aspirate (BAS, (C)). Horizontal green dotted lines represent the signal above 3 times the standard deviation of the blank. Error bars are the standard deviation (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Detection of IL-6 in real samples; (A) blood samples from healthy donors (black) or COVID-19 patients with IL-6 levels below (blue) or above (red) 17 pg·mL⁻¹; (B) Bronchial aspirate (BAS) samples from COVID-19 patients with IL-6 levels below 10 pg·mL⁻¹ (black), between 10 and 100 pg·mL⁻¹ (blue), or above 100 pg·mL⁻¹(red). Dotted lines show the average signal plus 2 times the standard deviation of healthy donors (A) or patients with IL-6 below 3 pg·mL⁻¹ (B). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)