key: cord-0130088-apahj1zn
authors: Zhang, Xiaoyan; Qi, Qige; Jing, Qiushi; Ao, Shen; Zhang, Zhihong; Ding, Mingchao; Wu, Muhong; Liu, Kaihui; Wang, Weipeng; Ling, Yunhan; Zhang, Zhengjun; Fu, Wangyang
title: Electrical probing of COVID-19 spike protein receptor binding domain via a graphene field-effect transistor
date: 2020-03-27
journal: nan
DOI: nan
sha: 2ae5bf9843256379921ddd22603012106322f1f7
doc_id: 130088
cord_uid: apahj1zn

Here, in an effort towards facile and fast screening/diagnosis of novel coronavirus disease 2019 (COVID-19), we combined the unprecedently sensitive graphene field-effect transistor (Gr-FET) with highly selective antibody-antigen interaction to develop a coronavirus immunosensor. The Gr-FET immunosensors can rapidly identify (about 2 mins) and accurately capture the COVID-19 spike protein S1 (which contains a receptor binding domain, RBD) at a limit of detection down to 0.2 pM, in a real-time and label-free manner. Further results ensure that the Gr-FET immunosensors can be promisingly applied to screen for high-affinity antibodies (with binding constant up to 2*10^11 M^-1 against the RBD) at concentrations down to 0.1 pM. Thus, our developed electrical Gr-FET immunosensors provide an appealing alternative to address the early screening/diagnosis as well as the analysis and rational design of neutralizing-antibody locking methods of this ongoing public health crisis.

Rapid and accurate identification/characterization of a potential pathogen is crucial for disease control, patient treatment and prevention of epidemic of emerging infectious diseases, such as the severe acute respiratory syndrome coronavirus (SARS-COV), which has incurred pandemics of respiratory infectious diseases with high mortality.

Recently, another emerging coronavirus that can cause viral pneumonia is outbreak in Wuhan, China and has propagated across the entire China. Meanwhile it has already shown great potential to have globe spread and thread to the worldwide public health. 1, 2 To date, nucleic acid-based molecular diagnostic tests based on the reverse transcription-polymerase chain reaction (RT-PCR), 3 have been established and widely adopted to identify this COVID-19. However, these detection methods require sophisticated primer and probe design, multi-step reactions, many reagents, trained personnel and bulky instruments. Moreover, it is impossible or inaccurate to detect infected but recovered people or asymptomatic carriers. In this respect, simple and costefficient protein-based immunosensor with high sensitivity is of vital importance when pestilential blast.

Graphene field-effect transistors (Gr-FET) are very attractive for the development of immunosensors due to their unprecedented sensitivity and chemical stability with proved capability for label-free digital biomolecules detection. [4] [5] [6] Compared to optical based immunoassays including fluorescence-linked and/or enzyme-linked immunosorbent assay (FLISA and/or ELISA) technology, Gr-FET biosensors eliminate the complicated procedure for fluorescence or enzyme labeling and do not require bulky and expensive optical instruments. Thus, facile Gr-FET biosensors with high sensitivity hold great potential for rapid diagnosis and early risk prediction of infectious diseases, which often present low copy numbers in vivo pathogen. In the case of coronaviruses, it has been reported that its spike (S) glycoprotein plays an extremely important role in recognizing the cell surface receptor, which is essential for both host specificity and viral infectivity. Further studies confirms that COVID-19 infects the human respiratory epithelial cells through the S1 subunit protein, which mainly contains a receptor binding domain (RBD) interacting with the human angiotensin-converting enzyme 2 (ACE2). 7 Moreover, the S protein also plays key parts in the induction of neutralizing-antibody and T-cell responses, as well as protective immunity. 8 Here, to enable digital detection of the virion attachment protein of coronaviruses for fast screening/diagnosis, we combined the extremely sensitive Gr-FET with highly selective SARS-COV spike S1 subunit protein antibody (CSAb) -COVID-19 spike S1 subunit protein (which contains the RBD) antigen interaction to develop an immunosensor. Strikingly, CSAb modified Gr-FET is capable of real-time detecting S1 at a limit of detection (LOM) down to 0.2 pM concentration with a fast responding time within 2 mins. Moreover, our results indicate that CSAb modified Gr-FET possesses a higher sensitivity than its counterpart with ACE2 receptors, which can be attributed to the higher bonding affinity of CSAb to S1 (K=2×10 11 M -1 ) compared to that of ACE2 (K=10 9 M -1 ). Thus, along with the capability of utilizing S1 modified Gr-FET to screening antibodies, we expand the potential utility of Gr-FET for rapid screening/diagnosis of respiratory infections caused by coronaviruses and its fast drug screening.

We started with high-quality monolayer single crystal graphene synthesized on single crystal Cu(111) foils by a chemical vapor deposition (CVD) method (Fig. S1 ), 9 and fabricated high-performance, solution-compatible Gr-FETs following an "upsidedown" process described previously. 10 Figure 1a depicts the schematics of Gr-FET for coronaviruses diagnosis, where graphene surface was specifically functionalized with either CSAb or ACE2 receptor to bind the S1 subunit protein from COVID-19. The hybridization of the slightly positively charged S1 protein with CSAb/ACE2 receptors immobilized on the graphene surface, alters its conductance/resistance via field effect, which can be electrically read out. We note here that we applied a reference electrode in constant contact with the antigen buffer solution to fix its electrostatic potential (Vref) during antibody-antigen reaction, and to control the current flow in the graphene channel between the source and the drain electrodes. As CSAb is positively charged, while ACE2 is negatively charged in PBS buffer solution (pH=7.2), we applied a negative or a positive potential at graphene during the incubation process, respectively, to improve their immobilization on the sensor surface. We functionalize the graphene surface with either CSAb or ACE2 receptor, which have proved affinity for the S1 subunit protein (which contains the RBD) from coronaviruses. (b) Electrical source-drain sheet conductance G as a function of the reference potential Vref measured for the CSAb immobilized Gr-FET. A bipolar transfer curve is observed corresponding to different type of charge carriers that can continuously be tuned from holes (left) to electrons (right) with a Dirac point VDirac at minimum G.

Due to Debye screen in the 10 mM PBS buffer solution (Debye length=0.7 nm), the charged S1 subunit protein cannot approach the graphene surface close enough to induce a notable field-effect, as the expected size of the antibody is around 7-10 nm, 11 i.e., one order-of-magnitude larger than the Debye length of the buffer solution. To responses (ΔVref) monitored at varying concentrations of S1 ranging from 0.2 pM to 10 nM (added at time intervals of about 5 mins with increasing concentration). The decrease of ΔVref depending on the increasing concentration of S1 is consistent with the bonding of slightly positively charged molecules on the sensor surface. Such CSAb-S1 complex can be refreshed and reused with comparable performance after thoroughly rinsing with buffer solution (Fig. S3) . Additionally, repeating the same measurement when shifting the liquid-gate voltage from Vref=0 V (electron branch) to Vref=-0.2 V (hole branch), yields similar sensing behavior as expected (Fig. S4) . As a negative control, pure T20 modified Gr-FET was also tested (Fig. S5 ). Compared to the CSAb immobilized Gr-FET immunosensor, T20 passivated Gr-FET shows negligible sensing response to the target S1. Therefore, our results clearly suggest that Gr-FET signal output is specific to the immobilized CSAb-S1 complex. Strikingly, such CSAb immobilized Gr-FET immunosensors target S1 subunit protein of COVID-19 with a limit of detection (LOD) down to 0.2 pM, which rivals that of state-of-the-art ELISA technology but eliminating the requirements of complicated procedure for enzyme labeling or bulky/expensive optical instruments. 12, 13 We note here that there is still plenty of room for improving the LOD by innovative antibody design and/or optimal Gr-FET sensing scheme. In addition, the real-time monitoring of the ΔVref response ( Fig. 2a) indicates a fast response time (within 2 min) for the detection of S1, which competes the current fast FLISA and/or ELISA technologies. 13 Alternatively, ACE2 is an integral membrane protein served as functional receptor for the spike glycoprotein of human coronaviruses SARS and COVID-19 infections. We further examined the affinity of ACE2-functionalized Gr-FET in the presence of S1 subunit protein at varying concentrations (10 pM-1 µM). In contrast to CSAb, real-time measurement of ACE2-functionalized Gr-FET immunosensor (Fig. 2b) suggest that sharp increase/change of ΔVDirac happened only when 1 nM S1 was added.

Further affinity fitting based on a single-reaction model 10 (see also SI) in Fig. 1e give binding affinities of K=2×10 11 M -1 and K=10 9 M -1 for CSAb-S1 and ACE2-S1 interaction, respectively. Obviously, compared to the ACE2 modified Gr-FET, its CSAb modified counterpart exhibits a much higher affinity to the S1 subunit protein of COVID-19, 14, 15 which is in consistent with previous reports that the antibody can scavenging virus before they bonding to receptors.

Neutralizing antibodies are extraordinarily crucial in the development of vaccines and antibody drugs. Here, to screen the neutralizing antibody, we functionalized Gr-FET with S1 protein to differential its affinity to CSAb or ACE2 in real-time. The results indicate a sensing response to CSAb even at 0.1 pM concentration; (Fig. 2d) whereas clear and sharp response can be identified for ACE2 only after 1 nM (Fig. 2e) . In Fig. 1f , affinity fitting using our previously determined binding constants of K=2×10 11 M -1 and K=10 9 M -1 gives good description for CSAb-S1 and ACE2-S1 interactions, respectively, also confirming our previous results and conclusions on CSAb or ACE2 immobilized Gr-FET for S1 detection. Here we ascribe the slight deviations in the affinity fitting for CSAb-S1 interaction at 0.1 pM and 1 pM concentration (Fig. 2f) to possible ionic screening of the weakly charged CSAb with relatively large size. (f) Affinity fittings using previously obtained equilibrium constants of S1 immobilized Gr-FET for CSAb-S1 (red dot and red line fit) and ACE2-S1 (blue dot and blue line fit) interactions, respectively. 

The − 1 complex system can be expressed by the dissociation constant :

− 1 + ⇔ + 1 + , =

here v is the number of sites of each species, 1 + the activity of the positively charged 1 + at the sensor surface, the binding affinity. The active surface sites are either complex − 1 + , or free . The total number of these sites is:

For simplifying the calculation, we assume the and 1 carrier zero and e charges, respectively. The complex − 1 + generates the total surface charge:

Eq. S1-3 can be rewritten:

These surface charge σ0 are screened by the ions of the double layer, yielding a surface potential drop Ψ0 over the double-layer capacitance CDL:

Via the Boltzmann equation, we relate the (unknown) activity of the surface 1 + in Eq. S4 with the (known) activity of the bulk solution 1 + :

Therefore, we may derive an explicit relationship between the bulk 1 + activity 1 + and the surface potential Ψ0:

The mechanism can be modeled similarly in case of 2 − 1 complex system.

Electrical probing of COVID-19 spike protein receptor binding domain via a graphene field-effect transistor

Yunhan Ling b , Zhengjun Zhang b , Wangyang Fu a,b,* a State Key Laboratory of New Ceramics and Fine Processing

The authors acknowledge financial support from the National Natural Science 

All authors declare that they have no conflict of interest.

Xiaoyan