Biomimetic Virus-based Colourimetric Sensors
Many materials in nature change colours in response to stimuli, making them attractive for use as sensor platform. However, both natural materials and their synthetic analogues lack selectivity towards specific chemicals, and introducing such selectivity remains a challenge. Here we report the self-assembly of genetically engineered viruses (M13 phage) into target-specific, colourimetric biosensors. The sensors are composed of phage-bundle nanostructures and exhibit viewing-angle independent colour, similar to collagen structures in turkey skin. On exposure to various volatile organic chemicals, the structures rapidly swell and undergo distinct colour changes. Furthermore, sensors composed of phage displaying trinitrotoluene (TNT)-binding peptide motifs identified from a phage display selectively distinguish TNT down to 300âp.p.b. over similarly structured chemicals. Our tunable, colourimetric sensors can be useful for the detection of a variety of harmful toxicants and pathogens to protect human health and national security.Our group identified a consensus TNT-binding peptide sequence (WHWQ) using phage display with a commercially available 12mer linear peptide library (Ph.D.-12). To incorporate the TNT-binding peptide, we genetically engineered the M13 phage's major coat proteins (pVIII). The desired peptide sequences were inserted between the first and the sixth amino acids of the amino terminus of wild-type pVIII, replacing residues 2â5 (Ala--Pro to Ala-()-Pro). To incorporate the most stable phage to carry the consensus TNT-binding peptide (WHWQ) identified by phage display, we designed a partial library with sequence of the form ADP using the primer: 5â²-ATATATCTGCAGCCCGCAAAAGCG GCCTTTAACTCCC-3â² and the primer 5â²-GCTGTCTTTCGCTGC AGAGGGTG-3â² to linearize the vector (=A/C/G/T and =G/T). To incorporate the gene sequences, PCR amplification was performed using Phusion DNA Polymerase, two primers (insertion and linearization) and an M13KE vector with an engineered I site as the template. The obtained product was purified on an agarose gel, eluted by spin column purification, digested with I enzyme and recircularized by an overnight ligation at 16âÂ°C with T4 DNA ligase. The ligated DNA vector was transformed into XL1-Blue electroporation competent bacteria and the amplified plasmid sequence was verified at the University of California, Berkeley, DNA sequencing facility. The pVIII library was screened against TNT and the resulting sequences were tested for stability after large-scale amplification. The library member, ADDWHWQEGDP, was finally chosen to create our TNTâPhage litmus sensors. Alanine-substituted control phage (WAW, AHW and WHA) sequences were synthesized by site-directed mutagenesis of the WHW phage. Using similar genetic engineering approaches, we constructed 4E phage as a control. The constructed phages were amplified using bacterial cultures and purified through standard polyethylene glycol precipitation. The phage solution was further purified by filtration through 0.45âÎ¼m pore size membranes. To verify phage stability, DNA sequences were confirmed at each step of the amplification.We created the phage self-assembled colour band patterns using a simple pulling method. The colours of the assembled structures were varied by controlling the pulling speed between 20 and 80âÎ¼mâmin. We constructed a home-built phage deposition apparatus by modifying a syringe pump. We programmed software using C to control the motor speed (between 0.1âÎ¼mâmin and 30âmmâmin) through an RS232C cable. For preparing Phage litmus matrices, we used 6âmgâml 4E phage suspensions in Tris-buffered saline (12.5âmM Tris and 37.5âmM NaCl, pH 7.5) or 2.4âmgâml WHW-phage suspensions in DI water. A spectrum of coloured bands (each band was obtained at a different pulling speed) was clearly perceptible when the matrices were deposited on gold-coated Si wafers.Fresh turkey head samples were donated from a local turkey farm (Pitman Farms, Sanger, CA, USA). The heads were obtained through overnight delivery immediately after they were slaughtered. The fresh turkey skin samples were immediately taken and processed for optical microscopy and transmission electron microscopy. For histology, 0.5 Ã 1âcm turkey skin samples were soaked in 20% sucrose in PBS for 2âh, embedded in OCT Compound (Sakura, Torrance, CA, USA), cryosectioned at 5âÎ¼m thickness (Shandon Cryostat, Asheville, NC) and stained with Masson's trichrome. Images were collected using an IX71 Microscope (Olympus, Tokyo, Japan). Transmission electron microscopy samples were fixed with 2% glutaraldehyde in 0.1âM sodium cacodylate (pH 7.2) for 1âh, post-fixed with 1% osmium tetroxide in 0.1âM sodium cacodylate (pH 7.2) and rinsed three times with sodium cacodylate (pH 7.2). Dehydration was done using a graded ethanol series (20, 40, 60, 80, 100 and 100%) followed by step-wise infiltration with epon-araldite resin (two parts acetone/one part resin for 1âh, one part acetone/one part resin for 1âh, one part acetone/two parts resin for 1âh, 100% resin for 1âh, 100% resin overnight, 100% resin with benzyl dimethylamine (BDMA) for 1âh) and heat polymerized in a 60âÂ°C oven. Sample blocks were sectioned at 90ânm using a Leica EM UC6 microtome (Leica Microsystems Inc., Buffalo Grove, IL 60089, USA). Grids were stained with 2% uranyl acetate and Reynolds' lead citrate. Imaging was done using a FEI Tecnai 12 transmission electron microscope (FEI, Hillsboro, OR 97124, USA).AFM images were collected using an MFP3D AFM (Asylum Research, Santa Barbara, CA) and analysed using Igor Pro 6.0 (WaveMetrics, Inc., Lake Oswego, OR) and Asylum software package (Asylum Research). All images were taken in tapping mode with a tip spring constant of 2âNâm. The probe tips (Ted Pella, Inc., Redding, CA) were made of silicon with a 10-nm radius. The humidity experiments were carried out in a closed liquid cell. We injected a fixed quantity of DI water to control the humidity. FFT analysis of AFM cross-sectional height profiles was used to determine the periodicity of the self-assembled nanofilament structures within the Phage litmus matrices. Numerical computation of the Fourier transform was done with a 2D FFT algorithm in OriginPro 8 (Origin Lab Corp., Northampton, MA) and imageJ v1.44p (National Institutes of Health, USA). We calculated the Fourier power spectra expressed in spatial frequency (Î¼m) from the AFM images.Phage litmus matrices were illuminated by a white light source of a Xenon lamp (X-Cite, Exfo, Mississauga, Canada) and the reflected spectra were obtained using a fibre optic spectrophotometer (USB4000, Ocean Optics, Dunedin, FL) through a Y-shaped bifurcated optical fibre (). An optical fibre fixed on an ââ stage was positioned normal to the Phage litmus surface and perpendicular to the scanning direction. Reflectance was measured using a gold-coated Si wafer as a reference. The humidity experiments were performed in a closed glove box (Plas Labs, Inc., Lansing, MI). The humidity was controlled by DI water and monitored using a hygrometer (VWR International Inc., West Chester, PA).To characterize the reflectance spectra of the Phage litmus under omnidirectional illumination, we lit a box coated with aluminium foil with a lamp (Incandescent Light, 150âW, DWC Sylvania, USA; ). We characterized the reflectance spectra while rotating the substrate between 10Â° and 90Â° from horizontal. A gold-coated Si wafer was used as a reference.To characterize the swelling behaviour of the Phage litmus, we performed GISAXS experiments before and during exposure to humidity (3âml of DI water in a closed chamber). The GISAXS data were collected at the beamline 7.3.3 at the Advanced Light Source at Lawrence Berkeley National Laboratory. X-rays with a wavelength of 1.23984âÃ
(10âkeV) were used, and the scattering spectra were collected on an ADSC Quantum 4âÎ¼m CCD detector with an active area of 188 Ã 188âmm (2,304 Ã 2,304 pixels). The scattering profiles were obtained after a 60-s collection time by integrating the 2D scattering pattern. The sample to detector distance was 1.84791, m and the incidence angle was 0.14Â°. Line-averaged intensities were reported as versus , where =(4/) Ã sin(/2), was the wavelength of incident X-rays and was the scattering angle.A home-built sensing and analysis system was developed for real-time chemical sensing. The equipment setup consisted of a gas chamber with an optical opening where a digital microscope (Celestron LLC, Torrance, CA) was attached to monitor the colour of the Phage litmus. The chamber with the Phage litmus inside was positioned on top of a heat block to control the temperature of the chamber. A MATLAB programme (Mathworks Inc., Natick, MA) was run on a PC to control the camera settings, to perform real-time readout and processing of the captured images and to display the real-time RGB data. First, the automatic gain of the digital microscope was turned to manual mode and a fixed gain was retained to prevent unwanted automatic compensation of brightness. The number of regions of interest, usually matching the number of different colour bands on the Phage litmus, was input by keyboard. Next, the specific regions to compare were selected from the first image (reference image) by mouse input. The subsequent images were taken and saved according to a pre-set frame rate (usually every 5âs). The change of the average RGB values with respect to the reference image for each region of interest was calculated and displayed on a graph in real time. We provide typical code from which we performed our experiments in . The vapour-phase experiments were performed by injection of a volume of solvent needed to achieve 300âp.p.m. concentration into a small container inside the chamber through an inlet tube. For explosive exposure experiments, we put excess amounts of each explosive crystal (200âmg) in a sealed chamber (20âml) and controlled the vapour pressures by temperature. We obtained the concentration of all compounds based on the vapour pressure at each temperature with the assumption of vapour ideality. The vapour pressure of TNT, DNT and MNT was obtained from previously reported values. To collect the data at saturated conditions, we held the Phage litmus in the sealed chamber for 30âmin and then obtained corresponding sensing results. To test the specificity of our TNTâPhage litmus sensor towards interfering molecules, experiments were performed in mixed vapours of MNT, DNT and TNT. First, we put the small sealed chambers (1âml) containing excess amounts of each explosive crystal (200âmg) in a large chamber with the phage litmus. Next, we exposed the chambers to MNT, DNT and TNT vapours sequentially using needles to open each small chamber (). A similar setup was used to perform the same experiments in a background of ethanol vapour (). We performed the control experiments using a 4EâPhage litmus sensor ().Surface plasmon resonance analyses were performed using the Kretschmann optical configuration. A tungsten halogen lamp with a multiwavelength light source was used and a polarizer was positioned on the input path of the light for transverse magnetic fields. The prism coupler and the Phage litmus were mounted on an stage. We made an enclosed cell of 100âÎ¼l using polydimethylsiloxane (PDMS) moulds. Flow of solution to the cell was implemented using 1âmm internal diameter tube. We injected 1âml of solution into the cell at a flow rate of 50âÎ¼lâmin. The outflow from the cell was carried through to a reservoir. The reflected spectrum was measured by a fibre optic spectrometer (USB4000-ultraviolet visible, Ocean Optics) and data acquisition was performed using a homemade LabVIEW programme (LabVIEW 2009, National Instrument, Austin, TX). The surface plasmon resonance spectrum was calculated from linearly polarized light parallel/perpendicular to the incidence plane (TM/TE configuration).iColour analyser is an iOS 5 application software built using the Xcode programming language (Xcode 4.2, Apple Inc., Cupertino, CA) and designed for the iPhone (Apple Inc, compatible with the iPod and iPad). This software has been built to analyse colourimetric changes of the RGB components from a Phage litmus in a systematic manner using a handheld device (). As demonstrated in this paper, the RGB colour components and their changes from the Phage litmus can be linked to specific responses from target molecules. The iColour analyser workflow is constituted of different parts (), as follows:: A channel type (picker) is displayed to choose an analysis mode: single or double channel. Single-channel mode samples from one to five colour matrices of a Phage litmus labelled as S1âS5. The results display one to five different colour matrix RGB components in a bar graph and their values on an 8-bit level. Double-channel mode allows for colour comparison analysis between a reference Phage litmus (before exposure to chemical) and the sample Phage litmus (after exposure to chemical) through comparison between one and five matrices. The RGB values displayed will be the difference between the RGB values of the sample and the reference.: Once the user clicks on the single button of this application, the iPhone takes a picture of the Phage litmus. One can resize and crop the picture of the Phage litmus sample to precisely obtain the target area to analyse.: Once the picture is taken, its central area is cropped and displayed. The picture is converted into a matrix containing the RGB and intensity values of each pixel. The vector data from the iPhone digital camera CCD (Omnivision OV5650, Santa Clara, CA) is processed to get the average of the RGB values from 2,592 Ã 1,944 pixels over the different areas selected by the user. An analysis table displays the RGB component intensity values and their s.d. An analysis graph will display the RGB values obtained from the sample. The 'average' picture displays a synthesized picture made from either the average RGB values in the case of the single analysis mode, or the difference between the average RGB values of the Phage litmus and its reference in the case of the double comparison mode. After displaying the analysis data and the date and time, a screenshot of the interface is saved in the picture browser of the iPhone and can be transferred easily to any computer through e-mail. In this work, for display purposes, the colour ranges of images are expanded from 5- to 8âbits per colour (RGB colour ranges of 0â31 expanded to 0â255).