key: cord-0900158-e9s12qq0
authors: Asandei, Alina; Mereuta, Loredana; Schiopu, Irina; Park, Yoonkyung; Luchian, Tudor
title: Teaching an old dog new tricks: A lipid membrane‐based electric immunosensor for real‐time probing of the spike S(1) protein subunit from SARS‐CoV‐2
date: 2021-10-07
journal: Proteomics
DOI: 10.1002/pmic.202100047
sha: c584963a3c52bc135a6b7de0a18b13108bf66ed9
doc_id: 900158
cord_uid: e9s12qq0

Fast, cheap, and easy to implement point‐of‐care testing for various pathogens constituted a game changer in past years due to its potential for early disease diagnosis. Herein, we report on the proof‐of‐concept of a simple method enabling in vitro detection of a structural spike protein subunit from the SARS‐CoV‐2 (S(1)) in aqueous samples. At the core of this discovery lies the well‐known paradigm of monitoring the capacitive current across a reconstituted zwitterionic lipid membrane subjected to a periodic transmembrane potential, followed by the real‐time spectral analysis enabling the extraction of the second harmonic of the capacitive current. Subsequent changes in the amplitude of this harmonic recorded during lipid membrane–S(1) interactions were correlated with alterations induced in the inner membrane potential profile by the S(1) protein subunit adsorption, and were shown to be augmented by ionic strength, the presence of a specific monoclonal antibody designed against the S(1) subunit and the angiotensin‐converting enzyme 2 (ACE2) protein receptor, and uninhibited by the presence of other human serum proteins.

freed from the need of expensive equipment remains critical. For such tasks and based on the cited examples, the four structural proteins of SARS-CoV-2, namely the spike surface glycoprotein (S), small envelope protein (E), matrix protein (M), and nucleocapsid protein (N), as well as their gene sequences and antibodies, could be used as targets for detection. The coronavirus entry into host cells and subsequent human pathogenicity is mediated by its 150 kDa transmembrane spike (S), heavily N-linked glycosylated homotrimer glycoprotein, comprising two functional subunits responsible for binding to the host cell receptor (S 1 subunit) with the receptor-binding domain (RBD) and fusion of the viral and cellular membranes (S 2 subunit) [4] [5] [6] . It is also known that for cell entry, SARS-CoV-2 specifically targets the transmembrane human angiotensin-converting enzyme 2 (ACE2) [7, 8] , with an affinity in the low nanomolar range [9] . As a viable alternative to the detection methods noted above, electrochemical biosensing platforms comprising bio-recognition elements (e.g., specific antibodies) and signal transducers enabled the direct detection of various respiratory viruses [10] including SARS-CoV-2 [11] .

In a recent effort made from our group toward with the established nanopore system [12, 13] , we showed that the S 1 subunit of the SARS-CoV-2 spike protein attaches to and permeabilizes lipid bilayer membranes even in the absence of a specific receptor protein [14] .

Inspired from previous work on membrane-active peptides [15, 16] , we hypothesized that prior to permeabilization, the lipid membrane adsorption of the S 1 subunit from the SARS-CoV-2 spike protein alters the distribution of mobile ions, surface, and polarization charges on the lipid membrane, all leading to changes in the electrical features the membrane, for example, the surface and/or dipole potentials. Here, by exploiting the dependence of the lipid membrane capacitance upon the potential difference across its hydrophobic core, we demonstrate that such changes are readily seen through monitoring the capacitive current across the lipid membrane substrate. Finally, we assemble our findings and propose a deceivingly simple, yet effective biosensing platform that allows for direct detection and recognition of the S 1 protein, based on its specific interaction with a designed monoclonal antibody and the ACE2 receptor, through the response elicited by such complexes on the membrane potentials of a reconstituted planar lipid membrane.

The specific SARS-CoV 2 proteins used herein were SARS-CoV- 

-A simple method enabling in-vitro detection of a structural spike protein subunit from the SARS-CoV-2 (S1) in aqueous samples is highly desirable.

-The amplitude of the second harmonic of the capacitive current through a lipid membrane substrate subjected to a periodic potential difference is correlated with alterations induced in the membrane potential profile by the S1 protein subunit adsorption.

-Such changes are augmented by ionic strength, the presence of a specific monoclonal antibody designed against the S1 subunit and the human angiotensin-converting enzyme 2 protein receptor (ACE2), and enable S1 detection in the nanomolar range.

The symmetrical membrane system used herein was obtained from DPhPC lipids dissolved in HPLC-grade n-pentane (10 mg mL −1 ) as described before [14, 17] . In short, the lipid bilayer (BLM, bilayer lipid membrane) was formed on a pre-treated with 10% v/v hexadecane in highly purified n-pentane aperture of ∼120 μm diameter in a 25 μm 

As illustrated in Figure 1A and demonstrated previously [18, 19] , in assessing the physical response of a lipid membrane to an applied potential difference ( Figure 1B If for a such model lipid membrane, the externally applied potential difference consists of a constant term (u 0 ) and a sinusoidal component with amplitude (u 1 ) and pulsation (ω) (ΔV ext = u 0 + u 1 sin( t)), by virtue of elementary circuit analysis, it follows that the resulting time-dependent capacitive current (I C (t)) embodies three harmonics of the fundamental pulsation (ω) (Supporting Information). For our analysis we focused solely on the second harmonic isolated from the power-spectra of the capacitive current (Figure 2A) , which in the time-domain and with the notations employed above writes: I 2 (t) = 3 sin(2t )C 0 ( + u 0 )u 2 1 . In this expression, the term δ stands for the lumped (ΔV S + ΔΔV D ) value quantifying the asymmetry in the surface and dipole potential of the lipid membrane (Supporting Informa-F I G U R E 2 (A) Typical representation of the power-spectra data on the capacitive current (I C ) measured in the "open air" (no lipid membrane formed) and respectively after the successful formation of a lipid membrane, subjected to a periodic ΔV ext with zero dc bias (u 0 ) (ΔV ext = u 1 sin(2 t); = 420 Hz and u 1 = 50 mV). In the latter case ("lipid membrane formed"), besides the fundamental harmonic measured at 420 Hz, the spectral analysis reveals the presence of two supplementary harmonics measured at 840 Hz (peak denoted herein by I 2 ) and 1260 Hz (peak denoted herein by I 3 ) (see the zoomed-in inset, the dashed encircled areas). The fact that the second harmonic (I 2 ) is non-zero at zero dc bias (u 0 = 0) (Supporting Information, "Theoretical account for the electric response of an elastic lipid membrane bilayer"), reflects among others an existing offset between the Ag/AgCl electrodes potentials, equivalent to applying a distinct from nil potential difference across the membrane. (B) The power-spectrum amplitude changes of the second harmonic (I 2 ) from the capacitive current (I C ) measured across a lipid membrane subjected to a periodic ΔV ext (ΔV ext = u 0 + u 1 sin(2 t); = 420 Hz and u 1 = 50 mV) with variable dc bias (u 0 ) (at u 0 = −50, −100, and −150 mV, the percent increase of I 2 was 25%, 140%, and 308%, respectively), with dc bias u 0 = 0 but exposed asymmetrically (cis side only) to SDS (C) (440% increase of I 2 after addition SDS [25 μM] and respectively 740% after addition SDS [50 μM]), and at constant dc bias (u 0 = −150 mV) but exposed asymmetrically (cis side only) to human serum (HS 1%) (D). HS, human serum; SDS, sodium dodecyl sulfate tion). It then follows that if the adsorption of certain membrane active molecules induces changes determines an asymmetry of the surface or dipole potentials (or both), the analyte detection can be easily achieved 

In Figure 3 , we present typical traces reflecting changes in the amplitude of the I 2 across a reconstituted lipid membrane, following asymmetric addition (cis side only) of nM amounts of the SARS-CoV-2 S 1 subunit (S 1 ), which we already established that adsorbs to the membrane [14] . 

For probing the detection specificity, our strategy involved the use of specific molecular substrates for the S 1 subunit, namely a synthesized monoclonal antibody and the ACE2 receptor. As shown in Figure 3 , following incremental addition of S 1 and assessment of I 2 changes the final injection of a monoclonal antibody (S 1 antibody) as to achieve a 1:4 (S 1 -S 1 antibody) molar ratio, determined a further augmentation in the I 2 amplitude. In a versatility test, a similar effect was seen in other experiments carried out in similar conditions, where the MERS-CoV spike S 1 subunit (MERS S 1 ) was employed instead ( Figure   S1 ). One should note that such effects are solely attributable for the resulting S 1 -S 1 antibody complex generated in the buffer, as by itself the S 1 antibody does not generate any distinguishable change in the I 2 amplitude ( Figure S2 ). To rationalize the lack of effect generated by the S 1 antibody alone, one must recall that according to the manufacturer, To mimic clinical conditions, we assessed the sensing capacity of the system described herein in the presence of commercial human serum added into the recording chamber. This is a vital step for any possible extension of the presented system to a field-deployable biosensor, since real human serum samples contain up to 10 4 proteins [25] , which may target the lipid membrane and affect non-specifically the sensitivity and specificity of detection. As we discovered, S 1 protein detection via S 1 -S 1 antibody complex formation and I 2 monitoring was unaffected by the presence of other blood proteins, judged from the fact that supplementary addition of 1% HS did not interfere with the detection process ( Figure S3 ).

In another set of experiments aimed at testing the detection specificity, addition of the more affine ACE2 receptor at a molar ratio of 1:4 (S 1 -ACE2) resulted in an even better S 1 detection response ( Figure 4) .

To interpret such increases in the I 2 amplitude through the Gouy-Chapman theory correlating surface potential (V S ) changes with the adsorbed analyte-induced surface charge density (σ) modifications [20] , a paradox arises. Knowing that at pH 6.3 as used herein, S 1 antibody is almost devoid of electric charge whereas the ACE2 receptor protein is negatively charged (S 1 antibody's pI = 6.47, according to the manufacturer and ACE2 receptor's pI = 5.8, calculated with the EMBL-EBI search and sequence analysis tools APIs), S 1 -S 1 antibody and S 1 -ACE2 complexes are expected to bear an almost unchanged (S 1 -S 1 antibody) to a lesser positive charge (S 1 -ACE2) as compared to the free S 1 protein. By comparison to the situation when S 1 was present alone in the electrolyte, it is then expected that S 1 -ACE2 would alter to a lesser extent the membrane surface charge density upon adsorption or leave it largely unchanged (S 1 -S 1 antibody), thus entailing smaller to none changes in the resulting ΔV H and the recorded I 2 amplitude, respectively (Figure 1Ab ). This on the other hand is in stark contrast with the experimental findings presented above (Figures 3   and 4) . The apparent paradox is further deepened by another obser-vation, according to which I 2 amplitude changes recorded following membrane interaction with either the S 1 alone or in complexation with the S 1 antibody are augmented in high salt electrolytes ( Figure S4 ). This is again puzzling, as the mobile ions-induced screening effect of the membrane electrostatics becomes prevalent in salt-concentrated electrolytes. Namely, a high versus Low ionic strength buffer is expected to better shield changes in the surface potential and the ensuing intramembrane electric field caused by membrane adsorption of a similar number of charged analytes. In other words and contrary to our findings, the I 2 amplitude should in fact be augmented in a low salt electrolyte. At the present, we can only speculate with regard to the precise molecular mechanism through which such complexes alter the physical properties of the lipid membranes and in accord to our observations. A plausible explanation would be that S 1 -S 1 antibody and S 1 -ACE2 complexes adsorption to the membrane will also increase the membrane dipole potential of the lipid monolayer where they adsorb to (ΔV D(cis) , Figure 1Ab , red lines). This is not unexpected, as previous data with other membrane insoluble analytes have demonstrated such an effect [20] . Moreover, such an effect would be facilitated in high ionic strength electrolytes-as shown herein, since the enhanced salt screening of the net electric charge on the complexes would in fact determine a more efficient accumulation of the complexes at the membrane surface or intercalation into the lipid matrix.

On the longer run, more experiments with different selections of lipid membrane-forming lipids (e.g., charged lipids, cholesterol) and electrolyte pH may also shed more light into the physical mechanism(s) contributing to the effects seen, through additional possible changes involving membrane fluidity and packing.

In this report, we established a simple to operate and effective setup to specifically detect in a time-resolved manner the SARS-CoV-2 S 1 protein subunit in aqueous solution and presence of physiologically relevant molecules. The use of both monoclonal antibodies and the

COVID-19) -World Health Organization (n.d

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Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2

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Advancements in electrochemical biosensing for respiratory virus detection: A review

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Nanoscale probing of informational polymers with nanopores. Applications to amyloidogenic fragments, peptides, and DNA-PNA hybrids

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Interaction between amphiphilic peptides and phospholipid membranes.Current Opinion in Colloid and Interface Science

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Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties

A virtual instrumentation based protocol for the automated implementation of the inner field compensation method

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Membrane electrostatics

Measurements and implications of the membrane dipole potential

Channelforming activity of cecropins in lipid bilayers: Effect of agents modifying the membrane dipole potential

Modulation of Lipid bilayer interfacial dipole potential by phloretin, RH421, and 6-ketocholestanol as probed by gramicidin channel conductance

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The human plasma proteome

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ACE2 receptor, as specific binding substrates, augmented the S 1 detection. The method opens the perspective of fast and cheap detection of other S 1 proteins containing receptor-binding residues mutations, artificial or natural antibodies in the aqueous sample (e.g., IgG and IgM), testing for efficacy of therapeutics-directed inhibitors (e.g., peptides) to the S proteins of SARS-CoV or related viruses, quantification of the adsorption kinetics of the antigen-antibody (receptor) complexes with a lipid membrane substrate of variable and controllable composition, or enable alternatives for monitoring of the interaction of viral antigens with selected protein targets, relevant for the discovery of decoy therapeutic proteins [26] . Despite its simplicity, we stress that the presented approach may evolve as a promising alternative, complementing established serological techniques [3] . In this context, 

The authors declare no conflict of interest.

The author has provided the required Data Availability Statement, and if applicable, included functional and accurate links to said data therein ORCID Tudor Luchian https://orcid.org/0000-0002-9388-7266