Tuning Electrostatic Gating of Semiconducting Carbon Nanotubes by Controlling Protein Orientation in Biosensing Devices.

The ability to detect proteins through gating conductance by their unique surface electrostatic signature holds great potential for improving biosensing sensitivity and precision. Two challenges are: (1) defining the electrostatic surface of the incoming ligand protein presented to the conductive surface; (2) bridging the Debye gap to generate a measurable response. Herein, we report the construction of nanoscale protein-based sensing devices designed to present proteins in defined orientations; this allowed us to control the local electrostatic surface presented within the Debye length, and thus modulate the conductance gating effect upon binding incoming protein targets. Using a ß-lactamase binding protein (BLIP2) as the capture protein attached to carbon nanotube field effect transistors in different defined orientations. Device conductance had influence on binding TEM-1, an important ß-lactamase involved in antimicrobial resistance (AMR). Conductance increased or decreased depending on TEM-1 presenting either negative or positive local charge patches, demonstrating that local electrostatic properties, as opposed to protein net charge, act as the key driving force for electrostatic gating. This, in turn can, improve our ability to tune the gating of electrical biosensors toward optimized detection, including for AMR as outlined herein.

Tuning Electrostatic Gating of Semiconducting Carbon Nanotubes by Controlling Protein Orientation in Biosensing Devices.

The ability to detect proteins through gating conductance by their unique surface electrostatic signature holds great potential for improving biosensing sensitivity and precision. Two challenges are: (1) defining the electrostatic surface of the incoming ligand protein presented to the conductive surface; (2) bridging the Debye gap to generate a measurable response. Herein, we report the construction of nanoscale protein-based sensing devices designed to present proteins in defined orientations; this allowed us to control the local electrostatic surface presented within the Debye length, and thus modulate the conductance gating effect upon binding incoming protein targets. Using a ß-lactamase binding protein (BLIP2) as the capture protein attached to carbon nanotube field effect transistors in different defined orientations. Device conductance had influence on binding TEM-1, an important ß-lactamase involved in antimicrobial resistance (AMR). Conductance increased or decreased depending on TEM-1 presenting either negative or positive local charge patches, demonstrating that local electrostatic properties, as opposed to protein net charge, act as the key driving force for electrostatic gating. This, in turn can, improve our ability to tune the gating of electrical biosensors toward optimized detection, including for AMR as outlined herein.

Switching protein metalloporphyrin binding specificity by design from iron to fluorogenic zinc.

Metalloporphyrins play important roles in areas ranging from biology to nanoscience. Using computational design, we converted metalloporphyrin specificity of cytochrome b562 from iron to fluorogenic zinc. The new variant had a near total preference for zinc representing a switch in specificity, which greatly enhanced the negligible aqueous fluorescence of free ZnPP in vitro and in vivo.

Site-Specific Protein Photochemical Covalent Attachment to Carbon Nanotube Side Walls and Its Electronic Impact on Single Molecule Function.

Functional integration of proteins with carbon-based nanomaterials such as nanotubes holds great promise in emerging electronic and optoelectronic applications. Control over protein attachment poses a major challenge for consistent and useful device fabrication, especially when utilizing single/few molecule properties. Here, we exploit genetically encoded phenyl azide photochemistry to define the direct covalent attachment of four different proteins, including the fluorescent protein GFP and a ß-lactamase binding protein (BBP), to carbon nanotube side walls. AFM showed that on attachment BBP could still recognize and bind additional protein components. Single molecule fluorescence revealed that on attachment to SWCNTs function was retained and there was feedback to GFP in terms of fluorescence intensity and improved resistance to photobleaching; GFP is fluorescent for much longer on attachment. The site of attachment proved important in terms of electronic impact on GFP function, with the attachment site furthest from the chromophore having the larger effect on fluorescence. Our approach provides a versatile and general method for generating intimate protein-CNT hybrid bioconjugates. It can be potentially applied to any protein of choice; the attachment position and thus interface characteristics with the CNT can easily be changed by simply placing the phenyl azide chemistry at different residues by gene mutagenesis. Thus, our approach will allow consistent construction and modulate functional coupling through changing the protein attachment position.

Detection of biogenic amines in pet food ingredients by RP-HPLC with automated dansyl chloride derivatization.

The detection of biogenic amines is of significant interest to the food industry, as they can be used as indicators of food spoilage and they are potentially toxic. Because of their importance, there is a need for automated methods suitable for industry use that can detect a wide range of biogenic amines at sufficient levels for food analysis. In this work, optimized conditions for the automated determination of biogenic amines (histamine, putrescine, cadaverine, spermine, spermidine, tyramine, and tryptamine) derivatized with dansyl chloride are presented. Limits of detection below 0.2 ppm were achieved for seven biogenic amines and percentage recoveries were between 80 and 109% for the seven analytes spiked into meat meal samples. The method is simple and compared well to an existing method for the detection of biogenic amines in pet food ingredients.

The determination and characterisation of 2-naphthyloxycarbonyl chloride derivatised biogenic amines in pet foods.

The need to monitor biogenic amines levels is essential for many areas of the food industry for two main reasons: the caustic nature and potential toxicity of these amines, and the potential to use amine levels as markers for freshness and quality in foodstuffs. Optimised analysis conditions used for the determination of biogenic amines derivatised with 2-napthyloxycarbonyl chloride has been applied to different pet food samples to assess the effectiveness of this method for complex sample matrices. Further to this, the use of high-resolution mass spectrometry has enabled the previously unconfirmed derivatised form of seven biogenic amines to be established. The derivatised forms identified include as mono substituted (tryptamine and histamine), bisubstituted (putrescine, cadaverine and tyramine), trisubstituted (spermidine) and tetrasubstituted (spermine). The methodology of biogenic amine determination was performed successfully to a range of pet food products highlighting the applicability to a variety of complex sample matrices.