Voltammetric Immunosensor Assembled on Carbon-Pyrenyl Nanostructures for Clinical Diagnosis of Type of Diabetes

This report investigates for the first time stability, scalability, and reusability characteristics of a protein nano-bioreactor useful for green synthesis of fine chemicals in aqueous medium extracting maximum enzyme efficiency. Enzyme catalysts conjugated with magnetic nanomaterials allow easy product isolation after a reaction involving simple application of a magnetic field. In this study, we examined a biocatalytic system made of peroxidase-like myoglobin (Mb), as a model protein, to covalently conjugate with poly(acrylic acid) functionalized magnetic nanoparticles (MNPs, 100 nm hydrodynamic diameter) to examine the catalytic stability, scalability, and reusability features of this bioconjugate. Application of the conjugate was effective for electrochemical reduction of organic and inorganic peroxides, and for both peroxide-mediated and electrocatalytic oxidation of the protein substrate 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) with greater turnover rates and product yields than Mb prepared in solution or MNP alone. Mb-attached MNPs displayed extensive catalytic stability even after 4 months of storage compared to Mb present in solution. Five- and ten-fold scale up of MNPs in the bioconjugates resulted in two- and four-fold increases in protein-catalyzed oxidation products, respectively. Nearly 40% of the initial product was present even after four reuses, which is advantageous for synthesizing sufficient products with a minimal investment of precious enzymes. Thus, the results obtained in this study are highly significant in guiding cost-effective development and efficient multiple uses of enzyme catalysts for biocatalytic, electrocatalytic, and biosensing applications via magnetic nanomaterials conjugation.

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“…2A). 27 Fig. 2B shows a coated layer of Mb around MNPs that is absent in free MNPs, which confirms the attachment of Mb to MNPs.…”

Section: Resultssupporting

confidence: 58%

Stability, scalability, and reusability of a volume efficient biocatalytic system constructed on magnetic nanoparticles

Premaratne

Nerimetla

Matlock

et al. 2016

Catal. Sci. Technol.

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“…39− The Sauerbrey equation is used to calculate the mass bound on to the quartz crystal at a thousand times greater sensitivity compared to an electronic balance with 0.1 μg sensitivity. 42 …”

Section: Resultsmentioning

confidence: 99%

Measuring ultra-low levels of nucleotide biomarkers using quartz crystal microbalance and SPR microarray imaging methods: A comparative analysis

Premaratne

Mubarak

Senavirathna

et al. 2017

Sensors and Actuators B: Chemical

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Circulating serum nucleotide biomarkers are useful indicators for early diagnosis of cancer, respiratory illnesses, and other deadly diseases. In this work, we compared detection performances of a quartz crystal microbalance (QCM), which is a mass sensor, with that of a surface plasmon resonance (SPR) microarray for an oligonucleotide mimic of a microRNA-21 biomarker. A surface immobilized capture oligonucleotide probe was used to hybridize with the target oligonucleotide (i.e., the microRNA-21 mimic) to facilitate selective detection. To obtain ultra-low femtomolar (fM) detection sensitivity, gold nanoparticles (50 nm) were conjugated with the target oligonucleotide. We achieved detection limits of 28and 47 fM for the target oligonucleotide by the QCM and SPRi microarray, respectively. We also conducted sample recovery studies and performed matrix effect analysis. Although the QCM had a lower detection limit, the microarray approach offered better throughput for analysis of up to 16 samples. We confirmed that the designed assay was selective for the target oligonucleotide and did not show signals for the control oligonucleotide with five mismatch sites relative to the target sequence. Combination of the QCM and microarray methods that utilize the same assay chemistry on gold are useful for overcoming clinical sample matrix effects and achieving ultra-low detection of small nucleotide biomarkers with quantitative insights.

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“…29 The number of serum insulin molecules conjugated to MNPs was estimated by enzyme linked immunosorbent assay (serum insulin ELISA kit, Mercodia, Sweden) similar to our voltammetry sensor report. 14 The number of free antibody molecules before and after conjugation to MNP were 4.4 (±0.2) × 10 13 and 1.6 (±0.1) × 10 13 , respectively, which indicates about 63% reaction efficiency. Estimation of the amount of serum insulin bound to Ab (insulin)2 -MNP yielded ~2.7 (±0.1) × 10 10 serum insulin bound to Ab (insulin)2 -MNP (500 μ L of 50% serum solution with 100 pM insulin corresponding to 3.0 × 10 10 insulin molecules) [ N = 3 replicates].…”

Section: Resultsmentioning

confidence: 90%

“…13,14 A number of other analytical methods for detection of insulin such as radioimmunoassay (RIA), enzyme linked immunoassay (ELISA), chemiluminescence immunoassay, electrochemiluminescence, and column immunoassay have also been reported. 15,16 These methods have the advantage of offering low detection limit in the picomolar range but the major drawbacks of these methods are (1) larger sample volumes, (2) longer assay times, and (3) no feature of real time monitoring coupled with a sensitive array image output as in SPRi.…”

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confidence: 99%

Magnetic Optical Microarray Imager for Diagnosing Type of Diabetes in Clinical Blood Serum Samples

2016

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Due to rapidly rising rates of diabetes and prediabetic conditions worldwide and the associated lethal complications, it is imperative to devise new diagnostic tools that reliably and directly measure insulin levels in clinical samples. Herein, we report a simple and sensitive direct imaging of insulin levels in diabetic patient samples using a surface plasmon resonance microarray imager (SPRi). To enhance sensitivity, we utilized magnetic nanoparticles (MNPs) to capture insulin from serum samples either directly or via a capture antibody immobilized on MNPs. The insulin-captured nanoparticles were allowed to bind surface insulin-antibody for detection from pixel intensity increase using a charge coupled device (CCD) built-in with the SPRi. We have compared the analytical figures-of-merit of the SPRi immunoarray on detecting insulin prepared in various percentages of serum solutions. A four parameter logistic model was used to obtain the best fit of microarray responses with insulin concentration and indicated the cooperative binding of insulin–nanoparticle conjugates to surface antibody in both the buffer insulin and the serum insulin conjugates with MNPs. The cooperativity effect is attributed to the greater association of magnetic nanoparticle-bound insulin molecules with increasing concentration of insulin binding to surface antibody. This is the first report of an SPRi immunoarray to accomplish clinical diagnosis of diabetic and prediabetic conditions based on insulin levels with serum matrix effect analysis and comparison between direct and sandwich insulin assay formats.

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Voltammetric Immunosensor Assembled on Carbon-Pyrenyl Nanostructures for Clinical Diagnosis of Type of Diabetes

Cited by 54 publications

References 36 publications

Stability, scalability, and reusability of a volume efficient biocatalytic system constructed on magnetic nanoparticles

Stability, scalability, and reusability of a volume efficient biocatalytic system constructed on magnetic nanoparticles

Measuring ultra-low levels of nucleotide biomarkers using quartz crystal microbalance and SPR microarray imaging methods: A comparative analysis

Magnetic Optical Microarray Imager for Diagnosing Type of Diabetes in Clinical Blood Serum Samples

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