1. Skinned Phage as a Component of Biosensor Patrick Kanju 1, Iryna B. Sorokulova 1, Solomon Yilma 1, Jennifer C. Sykora 2, Charles D. Ellis 4, Alexander Samoylov 1, W. Charles Neely 2, Valery A. Petrenko 3 and Vitaly J. Vodyanoy 1 1 Department of Anatomy Physiology & Pharmacology, 2 Department of Chemistry, 3 Department of Pathobiology, 4 Department of Electrical Engineering, Auburn University, AL, USA ABSTRACT Two phages selected from libraries were specific for binding of two proteins,  -galactosidase and streptavidin. These ‘affinity-selected’ phages can bind their protein antigens, thus functionally mimicking antibodies. This idea is illustrated by ‘‘detection’’ of proteins with ‘‘landscape’’ phage displaying a dense array of peptide binders on the surface. Monolayers containing biotinylated phospholipid were transferred onto the gold surface of an acoustic wave sensor using the Langmuir- Blodgett technique. Biotinylated phage was coupled with the phospholipid via streptavidin intermediates by molecular self-assembling. Phage biosensors based on QCM device showed high specificity, selectivity and affinity in binding of  -galactosidase and streptavidin. Phage coat proteins were isolated from phage by using newly introduced “phage skinning” technique. Phage proteins produced stable monolayers on the water/air interface. The monolayers can withstand a surface pressure up to 50 mN/m. When phage proteins were reconstituted in phospholipid bilayers they formed discrete and stable ion channels of uniform size and large amplitude ~ 30 pS. These results show that landscape phages can be utilized as substitutes to antibodies, and they demonstrate many features that make them potentially valuable as prospective probes for detection of biological threat agents. INTRODUCTION We have produced a biosensors capable of detecting proteins,  -galactosidase and streptavidin. Three methods will be used to immobilize/capture ligands on the sensor surfaces: (1) Combined Langmuir-Blodgett (LB) / molecular assembling method. The method includes LB deposition of a biotinylated monolayer onto a sensor surface and non-LB, molecular self-assembling of a phage layer using biotin/streptavidin coupling. (2) Direct physical adsorption of phage to sensor surfaces and (3) Phage skinning method. The recognition molecule used in this method is an octapeptide, VPEGAFSS, that is displayed at the N-terminus of all 4000 copies of major coat protein pVIII on the filamentous phage particle. The phage was separated from a billion- clone landscape phage library using affinity selection. Immobilization of the recognition probes was achieved using the LB technique, where monolayers of the phage-derived product were transferred onto the surface of a piezoelectric quartz crystal. The resulting films provide precise control of the film thickness and architecture, and preserve the sensitivity and specific recognition properties of the molecules. This study provides the first report of methods for the rapid and efficient production of phage coat protein monolayers. When transferred onto solid substrates, the monolayers result in films that may have unexpected electrical and optical properties and may be used in the preparation of biosensors, nano-machines and nano-structures with specifically distributed mechanical, chemical and optical properties. This new technique allows the control of surface characteristics of the monolayers which may be conjugated with other chemical/biochemical molecules and compounds to provide a wide variety of surface binding characteristics. Over the past several years, Hagen Bayley and co-workers have developed an alternate ion channel-based sensing scheme that relies on analysis of the temporal fluctuations of ion channel currents. This approach is adapted from biological receptors, which convert chemical signals into currents in ion channels. In this work we demonstrate ion channels constructed by modular design from phage coat proteins. The movement of current through these channels can be registered by conventional methods. The ion channels are triggered/activated by specific peptides, strategically inserted into phage coat proteins. Since the small size and planar architecture of the ion channels allows them to become components of a microelectronic circuit, they can be used for the detection of proteins, toxins, viruses, bacteria, and ions. Data analysis: Data segments of 5 – 120 sec lengths were digitized at 100  s intervals and transferred to a computer as data files. The data files were later subjected to statistical analysis using the Clampfit module of pCLAMP 9.0 data analysis program (Axon Instruments) as wells as the Microcal Origin 6.0 data analysis and technical graphics program. The resulting data points were compiled to generate the graphs displayed. METHODS Formation of bioselective layers The phage (7b1) used in this project was selected from billion-clone landscape phage libraries against streptavidin. Its binding activity was confirmed in ELISA tests. To form the bioselective layers, the phage was first converted to spheroids via chloroform treatment. Then monolayers were formed from spheroids. Finally the monolayers were deposited onto the sensor surface by Langmuir-Blodgett (LB) technique. Surface area-surface pressure isotherms: Lipid monolayer surface properties and depositions were carried out using (LB) film balances (Fig. 1B and Fig. 3) KSV 2200 LB and KSV5000 (KSV-Chemicals, Finland). Monolayers of phage coat proteins were made by allowing the spheroid suspension (~200μl) to run down a wettable glass rod that was partially submersed into the subphase at a slow constant rate of approximately 100 μl/min. After spreading, the glass rod was removed and the monolayer was allowed to equilibrate for 10 minutes at 21 o C. The monolayer was then compressed at a rate of 30 mm/min (45 cm 2 /min). Elasticity of the monolayer was calculated via the following equation: Elasticity =-A(d Π/dA), where A is the area of the film and Π is surface pressure. Reconstitution of phage protein in lipid bilayers: Lipid bilayers containing pure 1,2-diphytanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids, Inc.) were formed on the tip of patch pipettes using the tip-dip technique (Fig. 2 middle panel). The bilayers were formed by the successive transfer of two lipid monolayers upon the tip of the patch pipette. A phage spheroid suspension was vortexed and sonicated together with 10  g phospholipid (in hexane) and 10  l of buffer solution in order to form liposomes. The emulsion was then carefully transferred (Fig. 2 top panel) to the surface (air-fluid interface) of the solution on the cis-side of the membrane. Incorporation of phage protein into the membrane was achieved by dipping the tip of the patch pipette into the emulsion. The membrane incorporated phage proteins were subjected to voltage clamp studies using a patch clamp apparatus (Fig 1A). Resulting single channel current fluctuations were filtered at 5 kHz and recorded on a computer hard drive for later analysis. The voltage clamp amplifier was interfaced to a computer with a DigiData 1200 (Axon Instruments) data acquisition interface. Reconstitution of phage protein on solid substrate: Monolayers of pure phage protein were formed as described above. The monolayer was allowed to equilibrate and stabilize for 10 minutes at 19  0.1  C. It was then compressed at a rate of 30 mm/min and a vertical film deposition was carried out at a rate of 4.5 mm/min and a constant surface pressure of 30 mN/m. Mixed lipid monolayers were transferred onto the surface of quartz crystals (Fig 2 bottom panel). AT-cut planar quartz crystals (Fig 1C). with a 5 MHz nominal oscillating frequency were supplied by Maxtek, Inc. (SantaFe Springs, CA). Circular gold electrodes were deposited on both sides of the crystal for the electrical connection to the oscillatory circuit. Measurements were carried out using a PM-700 Maxtek microbalance (Santa Fe Springs, CA) with a frequency resolution of 0.5 Hz at 5 MHz and a mass resolution of 10 ng/cm 2. Voltage output of the Maxtek device was recorded and analyzed using a standard personal computer, data acquisition card and software. After positioning the sensor into the holder for measurement, 800 μl of the test solution was gently pipetted onto the sensor surface and the voltage was recorded for 12 to 16 minutes. The voltage output from the Maxtek device is directly related to the resonance frequency of the quartz crystal sensor. Changes in the resonance frequency of the quartz crystal sensor were used to monitor the binding of polystyrene beads (Fig. 1D) that were coated with either streptavidin or bovine serum albumin (BSA). The beads were obtained from Bangs Labs, Inc. and had a diameter of ~1μm. Figure 1. Laboratory Setup. (A) monolayer / bilayer voltage-clamp system. (B) Langmuir-Blodgett device. (C) Crystal sensors. (D) Monolayers of phage proteins on QCM sensor bind streptavidin-coated beads. A B C Streptavidin-coated beads QCM Sensor D coat proteins Figure 8. Ion gate membrane electrode array cross-section (A) and arrangement (B). BA Polymer Silicon Ag/AgCl Ion Gate Gold Electrode Insulator Electrode Amp Electrolyte CONCLUSIONS  Stable monolayers can be made from the chloroform-denatured phage and effectively transferred onto solid substrates.  Acoustic wave measurements reveal binding that is dose dependent and specific.  The results of this work demonstrate the feasibility of a biosensor based on Langmuir-Blodgett monolayers of chloroform-denatured phage.  Phage coat protein can be inserted into lipid bilayers in transmembrane configuration and can form ion channels. ACKNOWLEDGMENTS This work is supported by grants MDA972-00-1-0011 and AF-FA9550-04-1-0047 Figure 3. Monolayer system. Figure 4. Monolayer properties. (A) Surface pressure-area isotherm of monolayer formed when 7b1 spheroid suspension was spread at the air/water interface. (B) Graph of elasticity versus surface pressure (  ) for the monolayer. RESULTS AB Figure 2. Techniques for reconstituting phage protein in lipid membranes. Monolayer Formation Lipid Bilayer Formation Add 5  l Phosphatidylcholine Magnetic Stirrer Add 10  l AmB + chol mixture Phage protein Incorporation monolayer formation from lipid vesicles Bacteriophage is a thread-like virus of bacteria (left). It can be modified by genetic engineering methods to introduce new unique structural entities that are displayed on the virion’s surface (right). Gene 7 protein Gene 9 protein Gene 3 protein Gene 6 protein Gene 8 protein Circular ssDNA 1 - monolayer 2 - monolayer on substrate 3 - subphase 4 - trough 5 - barrier 6 - substrate 1 - MEMBRANE 2 - NANOPORES 3 - SILICON 4 - O-RING 5 - KCl SOLUTION 6 - ELECTRODE 7 - VOLTAGE CLAMP 1 2 3 4 5 6 7 6 1 Figure 6. Amino acid sequence of pVIII protein and hypothetical schematic of the arrangement of coat proteins at the air/water interface. AIR WATER NN C C Hydrophobic domain of phage protein Transfer of pVIII from Air/Water Interface to Solid Substrate Transfer of monolayer onto solid substrate Spreading of monolayer at air/water interface Substrate under monolayer Substrate slowly lifted out of subphase SUBPHASE Binding peptides Phage proteins Complete Biosensor B amino acid # 24681012141618202224262830323436384042444648505254 Alpha Regions I Alpha Regions II Hydrophilicity Plot 0 +4.5 -4.5 Alpha, Aphipathic Regions Beta, Amphipathic Regions Flexible Regions Antigenic Index 0 +1.7 -1.7 AVPEGAFSSDPAKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKAS 55 - - + - - + ++ + A Figure 5. Phage coat sensor for proteins  -galactosidase and streptavidin, fabricated by molecular assembling + LB (A & B), physical adsorption (C), and phage skinning (D). Phage Skinning D Physical Adsorbtion C Molecular Assembling + LB Relative Signal A [ B-gal ], nM Y/1-Y B analyte phage lipid Schematic view of filamentous phage infection E Attachment of streptavidin- coated beads of ~ 1  m F Conversion phages into spheroids of ~ 40 nm G Figure 7. Phage coat proteins were isolated from phage by using newly introduced “phage skinning” technique. Phage proteins produced stable monolayers on the water/air interface. When phage proteins were reconstituted in phospholipid bilayers they formed discrete and stable ion channels. When channels were formed from the phage selected to bind streptavidin addition of this protein to solution blocks the channel activity. Traces of single-channel recordings of spheroids in the absence (A) and in the precence of streptavidin were performed at a holding of 80mV. Each trace is 2.5 sec with amplitudes of approximately 25pA. The respective amplitude histograms in the absence (C) and in the presence (D) of 5 nM streptavidin are shown. The dwell time of open and closed level in the absence of streptavidin are shown in E and G respectively. Similarly the dwell levels for open and closed state in the presence of streptavidin are show in F and H respectively. Integration of natural and synthetic ion channels with electronic platforms enables rapid, real time conversion of molecular recognition events into digitized electronic signals. Phage Skinning technique provides a target-sensitive and specific ion channels. We employ photoelectrochemical effect for optical addressability.