Fe³⁺/Fe²⁺ Mycobactin-Complex Electrochemistry as an Approach to Determine Mycobactin Levels in Urine

Mycobacterium acquire iron by producing siderophores called mycobactins, with an extremely high affinity complexation of Fe3+. The iron complex shows distinctive electrochemistry predicting 18 orders of magnitude greater affinity of the mycobactin for Fe3+ than Fe2+. A heterogeneous standard rate constant, ks of the order of 10−5 cm s−1 confirms quasi reversible electrochemistry and based on the equilibrium in the presence of excess solution Fe3+/Fe2+ the oxidation and reduction peaks for the siderophore complex could be calibrated for ferric mycobactin J (FeMJ). FeMJ spiked urine collected from cows showed a matrix effect on the current peak height. For example, 240±15 µM FeMJ was estimated in 600 µM spiked urine. However, in the presence of excess solution Fe(acac)3 the same sample yielded an estimated 580±25 µM FeMJ.This study was supported by the Tuberculosis ResearchSection of Clifton E. Barry III as part of the Intramural Research Program of the National Institute of Allergy andInfectious Diseases, U.S. National Institutes of Health in the form of a stipend for author NSM.This is the accepted manuscript of a paper published in Electroanalysis Special Issue: Mátrafüred 2014 – International Conference on Electrochemical Sensors Volume 27, Issue 3, pages 833–842, March 2015, DOI: 10.1002/elan.20140056

Gene to Diagnostic: Self immobilizing protein for silica microparticle biosensor, modelled with sarcosine oxidase.

A rational design approach is proposed for a multifunctional enzyme reagent for point-of-care diagnostics. The biomaterial reduces downstream isolation steps and eliminates immobilization coupling chemicals for integration in a diagnostic platform. Fusion con-structs combined the central functional assay protein (e.g. monomeric sarcosine oxidase, mSOx, horseradish peroxidase, HRP), a visualizing protein (e.g. mCherry) and an in-built immobilization peptide (e.g. R5). Monitoring protein expression in E.coli was facilitated by following the increase in mCherry fluorescence, which could be matched to a color card, indicating when good protein expression has occurred. The R5 peptide (SSKKSGSYSGSKGSKRRIL) provided inbuilt affinity for silica and an immobilization capability for a silica based diagnostic, without requiring additional chemical coupling reagents. Silica particles extracted from beach sand were used to collect protein from crude protein extract with 85-95% selective uptake. The silica immobilized R5 pro-teins were stable for more than 2 months at room temperature. The Km for the silica-R52-mCh-mSOx-R5-6H was 16.5±0.9mM (com-pared with 16.5±0.4 mM, 16.3±0.3 mM, and 16.1±0.4 mM for R52-mCh-mSOx-R5-6H, mSOx-R5-6H and mSOx-6H respectively in solution). The use of the “silica-enzymes” in sarcosine and peroxide assays was shown, and a design using particle sedimentation through the sample was examined. Using shadowgraphy and particle image velocimetry the particle trajectory through the sample was mapped and an hourglass design with a narrow waist shown to give good control of particle position. The hourglass biosensor was demonstrated for sarcosine assay in the clinically useful range of 2.5 to 10 µM in both a dynamic and end point measurement regime.Royal Society IC160089 The Gates Cambridge Trust The Cambridge Trus

A molecular biology approach to protein coupling at a biosensor interface

Amino acid residues on the outside of proteins are discussed as potential sites for chemical coupling of proteins to sensor surfaces. This strategy is compared with the use of peptide tags, added to proteins, with an affinity for a particular surface material or chemical structure. Using molecular biology to extend the amino acid protein sequence, in order to include an immobilisation component, is also shown to be suitable for fusion to binding proteins, that can act as the immobilisation partner, so that a compendium of immobilisation strategies is seen to emerge from this common approach of protein engineering.This is the author accepted manuscript. The final version is available from Elsevier via https://doi.org/10.1016/j.trac.2016.01.02

Towards sarcosine determination in urine for prostatic carcinoma detection

Sarcosine, a potential biomarker for prostate cancer, can be detected in a solid state enzyme based biosensor using sarcosine oxidase, with particle immobilised reagents. A novel fusion protein of the fluorescent protein, mCherry, sarcosine oxidase (SOx), and the polypeptide R5 (R52-mCherry-SOx-R5-6H), was explored, which allowed self-immobilization on silica microparticles and long-term (90 days +) retention of activity, even at room temperature. In contrast, commercial wildtype SOx lost activity in a few days. A silica-R52-mCherry-SOx-R5-6H microparticle sensor for determination of sarcosine in urine, linked the SOx coproduct, H2O2, to a measurement catalysed by horseradish peroxidase (HRP) immobilised on silica, in the presence of Amplex Ultrared (AR) to generate fluorescence at 582 nm. Silica microparticles carrying all the reagents (R52-mCherry-SOx-R5-6H, HRP and AR) were used to produce a silica-microparticle biosensor which responded to sarcosine at micromolar levels. Interference by amino acids and uric acid was examined and it was found that the silica-reagent carrying system could be calibrated in urine and responded across the clinically relevant concentration range. This contrasted with similar assays using commercial SOx, where interference inhibited the sarcosine signal measurement in urine. The microparticle biosensor was tested in urine from healthy volunteers and prostate cancer patients, showing higher concentrations of sarcosine in cancer patients consistent with previous reports of elevated sarcosine levels.BBSRC/EPSRC funded Grant No. BB/L014130/1 Gates Cambridge Trust Generalitat Valenciana and European commission for its postdoctoral grant (APOSTD/113/2016) Generalitat Valenciana (PROMETEO 2016/109

Mapping minimum reflection distribution of surface plasmon resonance with a complex refractive index

One of the main challenges in making any further advances in the field of SPR biosensing is in the detection of small molecules, low analyte concentrations, and single particle interactions. To this end, labels have been used to amplify the signal of this traditional label-free technique. The physical properties associated with these labels can enhance the SPR signal by affecting the dielectric constant both in the real and imaginary parts. In this paper, we have taken a holistic approach to considering the dielectric properties of these labels and their effect on the minimum resonance intensity (MI) is mapped for the Kretschmann SPR configuration. These maps provide a way for matching label properties for particular SPR conditions and reveal that thicker Au films (∼65 nm) make the MI method more suitable for the detection of absorbing materials. Whereas the wavelength SPR at 50 nm Au film thickness generates a shift of similar or lower magnitude for adsorption of the absorbing label, compared with BSA adsorption, in the 'thick'-film SPR using the MI format, the signal due to the non-absorbing BSA adsorption tends towards zero (background), whereas the absorbing label produces a large well resolved signal.This research is supported by National Natural Science Foundation of China (NSFC, 31271064), International Exchanges Scheme 2012 China Costshare between Royal Society, UK and NSFC (31311130124), and Fundamental Research Funds for the central University

Functional Silver-Coated Colloidosomes as Targeted Carriers for Small Molecules

Colloidosomes have attracted great interest in recent years because of their capability for storage and delivery of small molecules for medical and pharmaceutical applications. However, traditional polymer shell colloidosomes leak low molecular weight drugs due to their intrinsic shell permeability. Here, we report aqueous core colloidosomes with a silver shell, which seals the core and makes the shell impermeable. The silver-coated colloidosomes were prepared by reacting l-ascorbic acid in the microcapsule core with silver nitrate in the wash solution. The silver shell colloidosomes were then modified by using 4,4′-dithiodibutyric acid and cross-linked with rabbit Immunoglobulin G (IgG). Label-free surface plasmon resonance was used to test the specific targeting of the functional silver shell with rabbit antigen. To break the shells, ultrasound treatment was used. The results demonstrate that a new type of functional silver-coated colloidosome with immunoassay targeting, nonpermeability, and ultrasound sensitivity could be applied to many medical applications.Qian Sun, Ziyan Zhao, and Hui Gao are grateful to the China Scholarship Council for funding. Yao Du is grateful to the Agency for Science Technology and Research (A*STAR) Singapore for funding

Microfluidics-based acoustic microbubble biosensor

This paper proposes a chip-scale microbubble-based biosensing platform. An encapsulated microbubble oscillates acoustically in liquid when exposed to an ultrasound field with its resonant frequency set by shell parameters. Changes in the resonant frequency of the microbubble can be used to monitor analyte-binding events on the shell. A device concept is proposed where ultrasonic transducers are integrated within a microfluidic channel, inside which electrodes are patterned for differential measurements of microbubble impedance. This device enables simultaneous measurements of the acoustic and electrical response of the microbubble, from which both mechanical and electrical parameters can be extracted. These parameters are used to provide a signature of the analyte. This paper presents acoustic and electrical models of the microbubbles, with the effect of shell parameters being thoroughly discussed. © 2013 IEEE

Capacitive touchscreen sensing - A measure of electrolyte conductivity

Mobile technologies such as smartphones and tablets combine computational power with inbuilt sensors and networking capabilities, making them ideal measurement instruments. There is already a rich history of research and commercially manufactured accessories taking advantage of their sensing and data visualisation capabilities. However, to-date the touchscreen component has not yet been translated to the widely established fields of capacitance-based bio- and environmental sensing. Here, we demonstrate the concept of contactless conductivity sensing of fluid samples placed directly on top of a projected mutual capacitive touchscreen with the measurement of a variety of electrolytes, leveraging the touchscreen's multi-touch capabilities. Electrolyte ions are particularly susceptible to the electric fringe field induced by capacitive touchscreens, and we report here a near-linear response to the ionic concentration of metal cations interesting for drinking water quality and soil health monitoring across a range of 0–500 μM (up to 100 μS). Simulation results are compared with experimental findings to reveal both the working principles and the key parameters that will be important for future sensing applications. This sensor demonstration is a starting point for broader exploration of the use of projected touchscreen sensing in mobile technologies and the creation of tools that are accessible to everyone, allowing rapid measurements and communication of data

Zein as biodegradable material for effective delivery of alkaline phosphatase and substrates in biokits and biosensors

A biodegradable material, zein, is proposed as a reagent delivery platform for biokits and biosensors based on alkaline phosphatase (ALP) activity/inhibition in the presence of phosphatase substrates. The immobilization and release of both the substrate and/or the active ALP, in a biodegradable and low-cost material such as zein, a prolamin from maize, and in combination with glycerol as plasticizer have been investigated. Three zein-based devices are proposed for several applications: (1) inorganic phosphorus estimation in water of different sources (river, lake, coastal water and tap water) with a detection limit of 0.2mg/L - compared to at least 1mg/L required by legislation, (2) estimation of ALP in saliva and (3) chlorpyrifos control in commercial preparations. The single-use kits developed are low cost, easy and fast to manufacture and are stable for at least 20 days at -20°C, so the zein film can preserve and deliver both the enzyme and substrates.The authors are grateful to Generalitat Valenciana (PROMETEO 2012/045 project and BEFPI/2014/014), Spain and to the Spanish Ministerio de Economía y Competitividad/FEDER (Project CTQ2014-53916-P). NJ expresses her gratitude to PROMETEO program for her predoctoral grant and BEFPI program for her stay grant