Surface Engineering of Graphene through Heterobifunctional Supramolecular-Covalent Scaffolds for Rapid COVID-19 Biomarker Detection

Abstract

Graphene is a two-dimensional semiconducting material whose application for diagnostics has been a real game-changer in terms of sensitivity and response time, variables of paramount importance to stop the COVID-19 spreading. Nevertheless, strategies for the modification of docking recognition and antifouling elements to obtain covalent-like stability without the disruption of the graphene band structure are still needed. In this work, we conducted surface engineering of graphene through heterofunctional supramolecular-covalent scaffolds based on vinylsulfonated-polyamines (PA-VS). In these scaffolds, one side binds graphene through multivalent π–π interactions with pyrene groups, and the other side presents vinylsulfonated pending groups that can be used for covalent binding. The construction of PA-VS scaffolds was demonstrated by spectroscopic ellipsometry, Raman spectroscopy, and contact angle measurements. The covalent binding of −SH, −NH2, or −OH groups was confirmed, and it evidenced great chemical versatility. After field-effect studies, we found that the PA-VS-based scaffolds do not disrupt the semiconducting properties of graphene. Moreover, the scaffolds were covalently modified with poly­(ethylene glycol) (PEG), which improved the resistance to nonspecific proteins by almost 7-fold compared to the widely used PEG-monopyrene approach. The attachment of recognition elements to PA-VS was optimized for concanavalin A (ConA), a model lectin with a high affinity to glycans. Lastly, the platform was implemented for the rapid, sensitive, and regenerable recognition of SARS-CoV-2 spike protein and human ferritin in lab-made samples. Those two are the target molecules of major importance for the rapid detection and monitoring of COVID-19-positive patients. For that purpose, monoclonal antibodies (mAbs) were bound to the scaffolds, resulting in a surface coverage of 436 ± 30 ng/cm2. K D affinity constants of 48.4 and 2.54 nM were obtained by surface plasmon resonance (SPR) spectroscopy for SARS-CoV-2 spike protein and human ferritin binding on these supramolecular scaffolds, respectively.

Biofunctionalization of Graphene‐Based FET Sensors through Heterobifunctional Nanoscaffolds: Technology Validation toward Rapid COVID‐19 Diagnostics and Monitoring

“…For the robust immobilization of affinity-proteins and antifouling polymers, surface engineering of GFETs through heterobifunctional supramolecular-covalent scaffolds based on VS-PEI (Figure 1F) was conducted as previously described. [33] Briefly, VS-PEI scaffolds were constructed onto GFETs by three simple surface modification steps: 1) the adsorption of pyrenebutanoic acid succinimidyl ester (PBSE) on graphene; 2) the fast reaction of polyethyleneimine (PEI) in aqueous solutions with surface-bound PBSE; and 3) the modification of remaining primary amine groups with divinylsulfone (DVS, a well-known cross-linker of SH, NH 2 , and OH groups via Michael type addition), [48][49][50][51] yielding VS-PEI on graphene. In these scaffolds, one side binds graphene through multivalent π-π interactions with pyrene groups, and the other side presents vinylsulfonated pending groups that can be used for covalent binding.…”

“…From these results it can be concluded that our functionalization approach does not disrupt the semiconducting properties of graphene, as previously demonstrated. [33] Next, recognition elements were bound to VS-PEI scaffolds prepared onto the graphene transistor. Three affinity-proteins were used as recognition elements: 1) Concanavalin A (ConA), a well-known lectin with high affinity to glycans; 2 a mAb specific to SARS-CoV-2 spike protein; 3) a mAb specific to human ferritin.…”

“…[29][30][31][32] In this regard, we have recently described the construction of vinylsulfonated-polyethyleneimine (VS-PEI) nanoscaffolds for performing the immobilization of both recognition and antifouling elements on graphene. [33] By employing this strategy, binding proteins and amino-terminated polyethyleneglycol (PEG) can be covalently bound to VS-PEI, while the whole architecture is attached to graphene by multivalent π-π interactions. Interestingly, the whole bio-interface was demonstrated to be more stable and presented better antifouling properties in comparison with that the obtained by a monopyrene-based approach.…”

“…Although there are some reports of sensing platforms prepared with Abs adsorbed onto PEI-modified graphenic surfaces [34][35][36][37] the VS-PEI nanoscaffold approach has the advantages of presenting covalent-like stability, even in strong surfactant solutions, making possible the sensor regeneration for reusing, and the capacity of anchoring both Abs and antifouling elements. [33] Compared with conventional SARS-CoV-2 antigen immunoassays, for example, ELISA and lateral-flow tests, [7,38] GFET sensing devices modified the heterofunctional VS-PA scaffolds displaying recognition and antifouling elements could have the following advantages: 1) simple digitization of the output data that could be used to improve medical record systems, that is, rapid access to information concerning a patient's health care; 2) GFET technology is fully compatible with the development of standalone smartphone-based biosensors; 3) only one type of recognition element (e.g., a capture Ab) is needed, whereas antigen immunoassay tests require the use of capture Abs, detector Abs, and nanoparticles or enzymes for the readout; 4) GFET test methodology is simpler and faster than ELISA since it does not require washing steps, incubation in solution with enzyme linked to detector Ab, enzymatic reaction, and a subsequent readout step with an additional laboratory equipment; 5) the activity of the Ab bound to VS−PEI on graphene could be restored with regeneration solutions, [33] a feature that is not accessible with lateral-flow tests, but may be particularly beneficial for reusing at-home antigen tests.…”

The Potential Contribution of Biopolymeric Particles in Lung Tissue Regeneration of COVID-19 Patients

“…These scaffolds offer vinylsulfonated awaiting groups for covalent attachment on one side and multivalent pyrene groups on the other. Construction of PA-VS scaffolds was shown using contact angle measurements and Raman spectroscopy [108]. Soft and hard tissue repair and regeneration, as well as wound healing, are possible using electrospun nanofiber scaffolds.…”