A colorimetric paper sensor for lactate assay using a cellulose-Binding recombinant enzyme

Abstract A new fabrication technology for effective preparation of paper-based biosensors using affinity-binding enzymes is developed. Lactate dehydrogenase (LDH) fused with a cellulose binding domain (CBD) is produced to allow one-step purification and immobilization of the enzyme on paper. Results demonstrate that the binding capacity of the recombinant enzyme is as high as 22 mg-enzyme/g-paper, with much improved stability (by a factor of 25-fold) in comparison to the native parent enzyme. That ensures the solid phase bioassay reactions on surface of the paper sensor are not limited by enzyme availability, and affords much extended sensor shelf life at the same time. The paper sensor shows a linear detection range of 0.5–8 mM of lactate, with changes in color intensity detectable within a much broader concentration range examined (up to 16 mM). The colorimetric display is suited for readouts using either human eyes or hand-held imaging devices such as smart phones. This work promises of a new strategy of developing high performance paper-based sensors for point-of-care diagnosis applications.

[1]  Xiaolian Gao,et al.  Expression of family 3 cellulose-binding module (CBM3) as an affinity tag for recombinant proteins in yeast , 2011, Applied Microbiology and Biotechnology.

[2]  Xinhao Ye,et al.  Quantitative determination of cellulose accessibility to cellulase based on adsorption of a nonhydrolytic fusion protein containing CBM and GFP with its applications. , 2007, Langmuir : the ACS journal of surfaces and colloids.

[3]  O. Shoseyov,et al.  Carbohydrate Binding Modules: Biochemical Properties and Novel Applications , 2006, Microbiology and Molecular Biology Reviews.

[4]  E. Fortunato,et al.  Office Paper Platform for Bioelectrochromic Detection of Electrochemically Active Bacteria using Tungsten Trioxide Nanoprobes , 2015, Scientific Reports.

[5]  M. Williams,et al.  Cloning of a lactate dehydrogenase gene from Clostridium acetobutylicum B643 and expression in Escherichia coli , 1990, Applied and environmental microbiology.

[6]  Dominic Rochefort,et al.  Confocal microscopy study of polymer microcapsules for enzyme immobilisation in paper substrates , 2009 .

[7]  Lauro T. Kubota,et al.  Amperometric biosensor for lactate based on lactate dehydrogenase and Meldola Blue coimmobilized on multi-wall carbon-nanotube , 2007 .

[8]  François Bertrand,et al.  Activity, stability and inhibition of a bioactive paper prepared by large-scale coating of laccase microcapsules , 2011 .

[9]  Y. Hadar,et al.  Oxidation of 4-bromophenol by the recombinant fused protein cellulose-binding domain-horseradish peroxidase immobilized on cellulose. , 2003, Biotechnology and bioengineering.

[10]  R. Bruccoleri,et al.  Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Bansi D. Malhotra,et al.  Co-immobilization of lactate oxidase and lactate dehydrogenase on conducting polyaniline films , 2000 .

[12]  E I Garvie,et al.  Bacterial lactate dehydrogenases. , 1980, Microbiological reviews.

[13]  Orawon Chailapakul,et al.  Use of multiple colorimetric indicators for paper-based microfluidic devices. , 2010, Analytica chimica acta.

[14]  Wouter Olthuis,et al.  Lactate biosensors: current status and outlook , 2013, Analytical and Bioanalytical Chemistry.

[15]  Patries M Herst,et al.  Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. , 2005, Biotechnology annual review.

[16]  Alberto J. Palma,et al.  A Portable Luminometer with a Disposable Electrochemiluminescent Biosensor for Lactate Determination , 2009, Sensors.

[17]  G. Brooks,et al.  Systemic lactate kinetics during graded exercise in man. , 1985, The American journal of physiology.

[18]  Jong Il Hong,et al.  Development of the smartphone-based colorimetry for multi-analyte sensing arrays. , 2014, Lab on a chip.

[19]  O. Kandler,et al.  Comparative studies of lactic acid dehydrogenases in lactic acid bacteria , 1977, Archives of Microbiology.

[20]  T. Lee,et al.  Evaluation of cellulose-binding domain fused to a lipase for the lipase immobilization , 2004, Biotechnology Letters.

[21]  G. Whitesides,et al.  Simple telemedicine for developing regions: camera phones and paper-based microfluidic devices for real-time, off-site diagnosis. , 2008, Analytical chemistry.

[22]  Bansi D. Malhotra,et al.  Immobilization of lactate dehydrogenase on electrochemically prepared polypyrrole–polyvinylsulphonate composite films for application to lactate biosensors , 2001 .

[23]  G. Whitesides,et al.  Patterned paper as a platform for inexpensive, low-volume, portable bioassays. , 2007, Angewandte Chemie.

[24]  Ali K. Yetisen,et al.  Applications of Paper-Based Diagnostics , 2015 .

[25]  M. Romero,et al.  Design and optimization of a lactate amperometric biosensor based on lactate oxidase cross-linked with polymeric matrixes , 2008 .

[26]  H. W. Doelle,et al.  Purification, properties and immunological relationship of L (+)-lactate dehydrogenase from Lactobacillus casei. , 1976, European journal of biochemistry.

[27]  Woo-Jin Chang,et al.  Paper-fluidic electrochemical biosensing platform with enzyme paper and enzymeless electrodes , 2014 .

[28]  N. Kotov,et al.  Simple, rapid, sensitive, and versatile SWNT-paper sensor for environmental toxin detection competitive with ELISA. , 2009, Nano letters (Print).

[29]  Orawon Chailapakul,et al.  Simple silver nanoparticle colorimetric sensing for copper by paper-based devices. , 2012, Talanta.

[30]  John D Brennan,et al.  Reagentless bidirectional lateral flow bioactive paper sensors for detection of pesticides in beverage and food samples. , 2009, Analytical chemistry.

[31]  Zhibin He,et al.  Development of cellulose paper testing strips for quick measurement of glucose using chromogen agent , 2012 .

[32]  Ping Wang,et al.  Peptide-induced affinity binding of carbonic anhydrase to carbon nanotubes. , 2015, Langmuir : the ACS journal of surfaces and colloids.

[33]  Minghui Yang,et al.  Paper based colorimetric biosensing platform utilizing cross-linked siloxane as probe. , 2014, Biosensors & bioelectronics.

[34]  G. Palazzo,et al.  Bioactive paper platform for colorimetric phenols detection , 2013 .

[35]  Babak A. Parviz,et al.  A contact lens with an integrated lactate sensor , 2012 .

[36]  Ping Wang,et al.  Printed microwells with highly stable thin-film enzyme coatings for point-of-care multiplex bioassay of blood samples. , 2015, The Analyst.