Detection of the Inflammatory Cytokine IL-6 in Complex Human Serum Samples Via Rational Nanotube Surface Passivation Screening
Authors: Gaikwad Pooja Vinod , Rahman Nazifa , Parikh Rooshi , Crespo Jalen , Cohen Zachary , Williams Ryan M. 
Published: September 2023

In recent years, biosensors have emerged as a tool with strong potential in medical diagnostics. Single-walled carbon nanotube (SWCNT) based optical nanosensors have notably garnered interest due to the unique characteristics of their near-infrared fluorescence emission, including tissue transparency, photostability, and various chiralities with discrete absorption and fluorescence emission bands.
The optoelectronic properties of SWCNT are sensitive to the surrounding environment, which makes them suitable for highly selective biosensing. Single-stranded (ss) DNA-wrapped SWCNTs have been reported as optical nanosensors for cancers and metabolic diseases. However, given the complexity of the human protein environment, non-specific interactions occur between SWCNT-based nanosensors and proteins. This inevitably leads to compromised selectivity of SWCNT-based nanosensors unless strategies for prevention are developed and employed.
Non-covalent passivation of the ssDNA-SWCNT surface is reported as an excellent strategy to improve nanosensor selectivity in complex biological settings without causing irreversible changes to the optical properties of SWCNTs. Emerging studies have explored and successfully shown passivation using proteins and phospholipids. However, a systematic comparative study of passivating agents has not been done. In this work, we explore and compare the efficacy of select proteins, polymers, and surfactants as passivating agents.
We, therefore, sought to evaluate various potential SWCNT surface passivation agents among broad classes of biomolecules and biomaterials. In the category of protein, Bovine serum albumin, dry-fat milk powder, and casein were selected due to their wide application to improve immunoassay selectivity. In the class of polymers, we selected 1 anionic and 2 cationic polymers, namely, polyethylene glycol, poly-ethylene imine, and poly-l-lysine. From surfactants, 2 phospholipids and 1 anionic surfactant: ammonium salt of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (16:0 PE2000PEG); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE PEG phospholipids), and sodium dodecylbenzene sulfonate (SDBS) were selected.
We analyzed the ability of all passivation agents to screen the non-specific interactions of ssDNA- SWCNTs in the presence of fetal bovine serum using fluorescence spectroscopy. The non-specific interactions between ssDNA-SWCNTs and proteins lead to a reorientation of the dipole moments and charge transfer in the corona phase around ssDNA-SWCNTs, leading to modulation in the center wavelength of the fluorescence as well as absorption peaks. We hence hypothesized that smaller changes in the center wavelength of the fluorescence peaks of passivated ssDNA-SWCNTs in presence of serum, lead to higher screening effect of the passivation agent towards non-specific interactions. We found that the most successful candidates were poly-L-lysine, polyethylene imine, dry-fat milk powder, and casein, in that order. Moreover, the ability to screen the interference was retained over a period of at least 3 hours. We further experimented with the four different mass ratios of passivating agents to ssDNA-SWCNTs and found that the mass ratio 50:1 for passivating agents: ss-DNA SWCNTs was most optimal.
We confirmed the strength of passivation using absorption spectroscopy. We hypothesized that stronger surface saturation of the SWCNT-TAT6 is by a passivating agent in buffer conditions leads to a larger shift in the absorption peaks. We found that for the above successful passivation agents, the order of passivation strength followed the same trend as the screening abilities of the successful passivating agents, supporting this mechanistic hypothesis.
Then, we evaluated an antibody-conjugated ssDNA-SWCNT nanosensor for the pro-inflammatory cytokine IL-6 with our successful passivation agents. We assessed the ability of the passivation agents to confer selective detection of IL-6 detection in clinical serum samples from patients with atherosclerosis and rheumatoid arthritis, both anticipated to have high IL-6 levels. Samples were compared to healthy human serum sample controls. We validated SWCNT fluorescence response with traditional immunoassays (ELISA).
We expect this study to provide rational strategies to screen interferences from non-specific interactions and improve the selectivity of the SWCNT-based optical nanosensors for in vivo applications.