A molecular biology approach to protein coupling at a biosensor interface

Hall, Elizabeth A. H.; Chen, Si; Chun, Jae Min; Du, Yao; Zhao, Ziyan

doi:10.1016/j.trac.2016.01.024

Cited by 11 publications

(7 citation statements)

References 74 publications

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“…Many affinity peptides have been identified for different materials [31] and in this example we have considered the use of silica as the core material platform for enzyme presentation. We have previously expressed a novel fusion protein: (R5)2-mCherry-sarcosine oxidase-R5-6H…”

Section: Resultsmentioning

confidence: 99%

“…However, it has been reported that immobilized enzymes can in some cases, exhibit higher selectivity and sensitivity [29], but on the other hand, chemical modification during immobilization can cause enzyme degradation or block the active centre, or result in the enzyme orientation being incorrect for reaction with the enzyme substrate. [30] Peptide molecular biology immobilization techniques can be inspired by high affinity peptides from binding patterns in nature [31] which allows an immobilisation functionality to be fused with the reagent enzyme (e.g. sarcosine oxidase) during protein expression.…”

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confidence: 99%

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Towards sarcosine determination in urine for prostatic carcinoma detection

Jornet-Martínez

Henderson

Campíns‐Falcó

et al. 2019

Sensors and Actuators B: Chemical

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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.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Towards sarcosine determination in urine for prostatic carcinoma detection

Jornet-Martínez

Henderson

Campíns‐Falcó

et al. 2019

Sensors and Actuators B: Chemical

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“…Since affinity peptides have been identified for an increasing range of substrates [79] including materials like cellulose and lignin, this approach has broad applicability to abundant naturally occurring materials. Such molecular and synthetic biology tools [80,81] may help reduce hurdles to local production in LMICs.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2019

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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 constructs 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 proteins 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 (compared 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.

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“…Thiol, amine, aldehyde, and carboxylic acid groups are likely also the best contestants in the future. With the increased research into binding peptides, , it would also be of interest to have those, biotin, or short peptide nucleic acid probes available as coupling groups on nanomaterials.…”

Section: Challenges and Future Potentialmentioning

confidence: 99%

Functional Nanomaterials and Nanostructures Enhancing Electrochemical Biosensors and Lab-on-a-Chip Performances: Recent Progress, Applications, and Future Perspective

et al. 2018

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Electrochemical biosensors and associated lab-on-a-chip devices are the analytical system of choice when rapid and on-site results are needed in medical diagnostics and food safety, for environmental protection, process control, wastewater treatment, and life sciences discovery research among many others. A premier example is the glucose sensor used by diabetic patients. Current research focuses on developing sensors for specific analytes in these application fields and addresses challenges that need to be solved before viable commercial products can be designed. These challenges typically include the lowering of the limit of detection, the integration of sample preparation into the device and hence analysis directly within a sample matrix, finding strategies for long-term in vivo use, etc. Today, functional nanomaterials are synthesized, investigated, and applied in electrochemical biosensors and lab-on-a-chip devices to assist in this endeavor. This review answers many questions around the nanomaterials used, their inherent properties and the chemistries they offer that are of interest to the analytical systems, and their roles in analytical applications in the past 5 years (2013−2018), and it gives a quantitative assessment of their positive effects on the analyses. Furthermore, to facilitate an insightful understanding on how functional nanomaterials can be beneficial and effectively implemented into electrochemical biosensor-based lab-on-a-chip devices, seminal studies discussing important fundamental knowledge regarding device fabrication and nanomaterials are comprehensively included here. The review ultimately gives answers to the ultimate question: "Are they really needed or can bulk materials accomplish the same?" Finally, challenges and future directions are also discussed.

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A molecular biology approach to protein coupling at a biosensor interface

Cited by 11 publications

References 74 publications

Towards sarcosine determination in urine for prostatic carcinoma detection

Towards sarcosine determination in urine for prostatic carcinoma detection

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

Functional Nanomaterials and Nanostructures Enhancing Electrochemical Biosensors and Lab-on-a-Chip Performances: Recent Progress, Applications, and Future Perspective

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