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. 2020 Oct 20;53(10):2384-2394.
doi: 10.1021/acs.accounts.0c00474. Epub 2020 Oct 1.

Viruses Masquerading as Antibodies in Biosensors: The Development of the Virus BioResistor

Apurva Bhasin  1 , Nicholas P Drago  1 , Sudipta Majumdar  1 , Emily C Sanders  1 , Gregory A Weiss  1   2   3 , Reginald M Penner  1
Affiliations

Viruses Masquerading as Antibodies in Biosensors: The Development of the Virus BioResistor

Apurva Bhasin et al. Acc Chem Res. .

Abstract

The 2018 Nobel Prize in Chemistry recognized in vitro evolution, including the development by George Smith and Gregory Winter of phage display, a technology for engineering the functional capabilities of antibodies into viruses. Such bacteriophages solve inherent problems with antibodies, including their high cost, thermal lability, and propensity to aggregate. While phage display accelerated the discovery of peptide and protein motifs for recognition and binding to proteins in a variety of applications, the development of biosensors using intact phage particles was largely unexplored in the early 2000s. Virus particles, 16.5 MDa in size and assembled from thousands of proteins, could not simply be substituted for antibodies in any existing biosensor architectures.Incorporating viruses into biosensors required us to answer several questions: What process will allow the incorporation of viruses into a functional bioaffinity layer? How can the binding of a protein disease marker to a virus particle be electrically transduced to produce a signal? Will the variable salt concentration of a bodily fluid interfere with electrical transduction? A completely new biosensor architecture and a new scheme for electrical transduction of the binding of molecules to viruses were required.This Account describes the highlights of a research program launched in 2006 that answered these questions. These efforts culminated in 2018 in the invention of a biosensor specifically designed to interface with virus particles: the Virus BioResistor (VBR). The VBR is a resistor consisting of a conductive polymer matrix in which M13 virus particles are entrained. The electrical impedance of this resistor, measured across 4 orders of magnitude in frequency, simultaneously measures the concentration of a target protein and the ionic conductivity of the medium in which the resistor is immersed. Large signal amplitudes coupled with the inherent simplicity of the VBR sensor design result in high signal-to-noise ratio (S/N > 100) and excellent sensor-to-sensor reproducibility. Using this new device, we have measured the urinary bladder cancer biomarker nucleic acid deglycase (DJ-1) in urine samples. This optimized VBR is characterized by extremely low sensor-to-sensor coefficients of variation in the range of 3-7% across the DJ-1 binding curve down to a limit of quantitation of 30 pM, encompassing 4 orders of magnitude in concentration.

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Figures

Figure 1 –
Figure 1 –
The covalent virus surface (CVS). a). Stepwise assembly (steps 1–3) and functionalization (steps 4–6) of the CVS. (b-d) Noncontact mode AFM images (1 μm × 1 μm). (b) A single M13 virion on mica, (c) A self-assembled monolayer (SAM) of N-hydroxy-succinimide thioctic ester on gold after exposure to BSA. No virus particles were attached to this surface. (d) A functional CVS consisting of a SAM of N-hydroxysuccinimide thioctic ester (NHS-TE) on gold, reacted with M13 to produce covalent attachment, and exposed to BSA (Figure 1, step 3). After Ref.
Figure 2 –
Figure 2 –
QCM investigations of the CVS. a,b) Schematic diagram of the QCM and flow cell. c). QCM evaluation of the efficacy of three wash solutions as indicated. d,e). d) Plot of mass versus time for the exposure of a CVS to doses of p8-Ab, ranging in concentration from 6.6 to 200 nM. e). Same data as shown in (d), but normalized to the same injection time to precisely show relative heights of these transients. f) Plot of maximum mass change versus p8-Ab concentration for the data shown in (d,e). The mass change was proportional to the concentrations of injected p8-Ab (R2 = 0.997) and yielded a sensitivity of 0.018 (μg cm−2)/nM and a limit of detection (LOD) of 6.6 nM. After Ref.
Figure 3 –
Figure 3 –
Electrodeposition of a virus-PEDOT bioaffinity layer. a). The virus-PEDOT electrodeposition reaction, b). QCM analysis of virus-PEDOT electrodeposition shows increased mass loading as a decrease in frequency. c) Frequency change versus deposition charge, Qtot, for QCM measurements. d) Calibration curve showing the linear correlation of the virus concentration within the PEDOT film (vertical axis) versus the concentration of virus in solution. (e-j). Topography of virus-PEDOT films imaged by scanning electron microscopy. Films were prepared from solutions containing virus particles at three concentrations: (e,f) [virus]soln = 3 nM, (g,h) [virus]soln = 9 nM, and (I,j) [virus]soln = 15 nM. After Ref.
Figure 4 –
Figure 4 –
PSMA Detection in Synthetic Urine Using Synergistic, Dual-Ligand Phage. a) Schematic diagram of bidentate binding to PSMA by KCS-1, (green) and genetically encoded peptide, (red). Simultaneous binding by these two ligands provides higher apparent affinity to PSMA. b). Polymerization reactions of EDOT in the presence of: (top) LiClO4 or (center)) PSMA-binding phage, and (bottom) PSMA-binding phage and exposure to the wrapper KCS-1 (Green), c). Schematic diagram of the biosensing experiment. (d) ΔR/Ro of the film increases with the PSMA concentration. e). Comparison of PSMA detection in synthetic urine (green) with detection in PBF buffer (purple). After Ref.
Figure 5 –
Figure 5 –
The Two-Sided Biosensor: A Monolithic Biosensor for Human Serum Albumin (HSA). a). Engineering diagram of two electrode virus-PEDOT biosensor. b,c) Nyquist plots (Zim vs. Zre) for a control protein (BSA) and HSA. d). Signal-to-noise versus frequency plot for HSA and BSA. e). ΔRre versus HSA concentration calibration curve. Controls for BSA, and off-virus binding also shown. After Ref.
Figure 6 –
Figure 6 –
The Virus BioResistor (VBR). a) VBRs are constructed on a 10 mm glass chip with patterned gold electrodes. b). Three processing steps provide for the deposition of a PEDOT-PSS layer by spin-coating from solution (Step 1), the attachment of a PMMA cell (Step 2), and the electrodeposition of a virus-PEDOT layer (Step 3). c). The electrical response of the VBR is modeled by three parallel resistors and a solution/channel capacitance. d). This circuit produces a semi-circular Nyquist plot for which the high frequency impedance (40 kHz) approximates the solution resistance (Zre ≈ Rsoln), and the low frequency impedance is dominated by the parallel resistance imposed of the two film resistors, Zre ≈ ZVBR. ZVBR increases with the concentration of target protein present in the solution phase. e). The VBR circuit maximizes signal-to-noise (S/N) at low frequencies, and can exceed 100 at high protein concentrations. After Ref.
Figure 7 –
Figure 7 –
Rapid Quantitation of DJ-1 in Urine. a,b) Correlation of VBR signal against DJ-1 concentration in urine and synthetic urine. c). Comparison of DJ-1 signal at 300 nM with three controls, d). VBR signal versus time for the exposure of five VBRs to aliquots of DJ-1 in synthetic urine. e,f). Proposed mechanism for VBR signal transduction. After Ref.
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References

    1. Yang LMC; Tam PY; Murray BJ; McIntire TM; Overstreet CM; Weiss GA; Penner RM Virus Electrodes for Universal Biodetection. Anal. Chem 2006, 78, 3265–3270.

      Our first biosensor exploiting immobilized virus particles, instead of antibodies, as receptors.

    1. Donavan KC; Arter JA; Weiss GA; Penner RM Virus-Poly(3,4-Ethylenedioxythiophene) Biocomposite Films. Langmuir 2012, 28, 12581–12587.

      First description and characterization of the electrodeposition of virus-PEDOT composite films.

    1. Bhasin A; Ogata AF; Briggs JS; Tam PY; Tan MX; Weiss GA; Penner RM The Virus Bioresistor: Wiring Virus Particles for the Direct, Label-Free Detection of Target Proteins. Nano Lett. 2018, 18, 3623–3629.

      The first paper describing the Virus BioResistor (VBR).

    1. Bhasin A; Sanders EC; Ziegler JM; Briggs JS; Drago NP; Attar AM; Santos AM; True MY; Ogata AF; Yoon D V; et al. Virus Bioresistor (VBR) for Detection of Bladder Cancer Marker DJ-1 in Urine at 10 PM in One Minute. Anal. Chem 2020, 92, 6654–6666.

      First demonstration of detection, at 10 pico-molar concentrations, of a cancer marker in human urine using the VBR.

    1. Smith GP; Petrenko VA Phage Display. Chem. Rev 1997, 97, 391–410. - PubMed

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