Biosensing with Virus Electrode Hybrids

Kritika Mohan1, Reginald M. Penner2, Gregory A. Weiss3

1 Department of Chemistry, University of California, Irvine, California, 2 Department of Chemical Engineering and Materials Science, University of California, Irvine, California, 3 Department of Molecular Biology and Biochemistry, University of California, Irvine, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch140213
Online Posting Date:  June, 2015
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Abstract

Virus electrodes address two major challenges associated with biosensing. First, the surface of the viruses can be readily tailored for specific, high affinity binding to targeted biomarkers. Second, the viruses are entrapped in a conducting polymer for electrical resistance‐based, quantitative measurement of biomarker concentration. To further enhance device sensitivity, two different ligands can be attached to the virus surface, and increase the apparent affinity for the biomarker. In the example presented here, the two ligands bind to the analyte in a bidentate binding mode with a chelate‐based avidity effect, and result in a 100 pM experimentally observed limit of detection for the cancer biomarker prostate‐specific membrane antigen. The approach does not require enzymatic amplification, and allows reagent‐free, real‐time measurements. This article presents general protocols for the development of such biosensors with modified viruses for the enhanced detection of arbitrary target proteins. © 2015 by John Wiley & Sons, Inc.

Keywords: phage display; phage wrapping; click chemistry; prostate‐specific membrane antigen; electrochemical impedance spectroscopy

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Click Chemistry Reaction: Preparation of KCS‐1 and KCS‐2
  • Basic Protocol 2: Phage Propagation, Isolation, Purification, and Quantification
  • Basic Protocol 3: Biosensing: Formation of the Bioaffinity Matrix and Electrochemical Detection
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Click Chemistry Reaction: Preparation of KCS‐1 and KCS‐2

  Materials
  • Alkyne‐functionalized peptide stock solution (200 μM in water)
  • Triethylammonium acetate buffer (1 M, pH 7)
  • HPLC‐grade water or Milli‐Q water
  • Azide‐functionalized peptide stock solution (200 μM in 1:3 water/acetonitrile mixture)
  • Ascorbic acid
  • Copper sulfate (CuSO 4)
  • Micro concentrator

Basic Protocol 2: Phage Propagation, Isolation, Purification, and Quantification

  Materials
  • E. coli XL1 Blue cells (CaCl 2 competent; e.g., Stratagene)
  • M13 phage‐display vectors (phagemids, Vrisko Limited)
  • 2YT medium (16 g tryptone, 10 g yeast extract, 5 g NaCl; adjust to 1 liter with Milli‐Q purified water, pH 7; sterilize by autoclaving)
  • LB agar plates
  • Carbenicillin stock (50 mg/ml in sterile water, i.e., autoclaved Milli‐Q purified water)
  • Tetracycline stock (5 mg/ml in sterile water, i.e., autoclaved Milli‐Q purified water)
  • Kanamycin stock (40 mg/ml in sterile water, i.e., autoclaved Milli‐Q purified water)
  • M13 KO7 helper phage (GE Healthcare Life sciences)
  • PEG‐NaCl (2.5 M NaCl, 20% PEG 8000)
  • PBS‐Tween (PBS: 135 mM NaCl, 2.5 mM KCl, 8 mM Na 2HPO 4, 30 mM KH 2PO 4, pH 7.2 with 0.05% Tween‐20 added)
  • 12 mM lithium perchlorate (LiClO 4) solution
  • 15‐ml Falcon tubes
  • 50‐ and 250‐ml centrifuge bottles
  • UV transparent plates (cat. no. 3635, Corning)
  • Microtiter plate reader (Bio‐Tek)
NOTE: All buffers and solutions used for phage propagation are sterilized prior to use by autoclaving. Solutions such as PBS‐Tween are sterile‐filtered after addition of Tween to sterile PBS.

Basic Protocol 3: Biosensing: Formation of the Bioaffinity Matrix and Electrochemical Detection

  Materials
  • Lithium perchlorate (LiClO 4)
  • Ethylene‐3,4‐dioxythiophene (EDOT)
  • Phage stocks (see protocol 2)
  • Phosphate‐buffered fluoride (PBF)‐Tween buffer (PBF: 4.2 mM Na 2HPO 4, 1.5 mM KH 2PO 4 and 140 mM NaF, pH 7.2; sterile filtered, with added 0.1% Tween 20)
  • K CS1 (see protocol 1; used as a solution: dissolve K CS1 in a 40:60 acetonitrile/HPLC‐grade water solution to a final concentration of 1 μg/μl; K CS1 stock can be stored indefinitely at −20°C)
  • Piranha solution (optional; sulfuric acid [H 2SO 4] and 30% H 2O 2)
  • 1‐, 0.5‐ and 0.25‐μm particle size diamond polishing paste (Ted Pella)
  • Circular gold (Au) electrode (CH Instruments)
  • Micropolishing cloth (Buehler)
  • Platinum counter electrode
  • Ag/AgCl reference electrode
  • Parstat 2273 potentiostat
  • POWERCV and POWERSine software (Princeton Applied Research)
  • Glass vial
  • Kimwipes
  • Butane torch
  • Faraday cage
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Figures

Videos

Literature Cited

Literature Cited
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Internet Resources
  http://www.lumiprobe.com/protocols/click‐chemistry‐dna‐labeling
  Lumiprobe protocol: Click‐Chemistry Labeling of Oligonucleotides and DNA.
  https://www.addgene.org/plasmid‐protocols/bacterial‐transformation/
  Addgene protocol: Heat‐shock transformation of plasmids/phagemids into chemically competent bacterial cells.
  https://www.addgene.org/plasmid‐protocols/bacterial‐transformation/
  Addgene protocol: Heat‐shock transformation of plasmids/phagemids into chemically competent bacterial cells.
  http://www.graphpad.com
  GraphPad Prism software has been used for biosensing data analysis. GraphPad software was founded by CEO Dr. H. J. Motulsky, located in San Diego, CA.
  http://www.virology.ws/2011/01/13/multiplicity‐of‐infection
  Prof. Vincent Racaniello, Ph.D., Higgins Professor of Microbiology & Immunology in the College of Physicians and Surgeons of Columbia University, discusses the concept of multiplicity of infection from a statistical standpoint.
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