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Hemoglobin Immobilization on Multiporous Nanofibers of SnO₂ and Chitosan Composite for Hydrogen Peroxide Sensing

Kafi AKM, Alim S, Jose R, Yusoff MM

J Nanosci Nanotechnol, 2019 04 01;19(4):2027-2033.
PMID: 30486943 DOI: 10.1166/jnn.2019.15465

A multiporous nanofiber (MPNFs) of SnO₂ and chitosan has been used for the immobilization of a redox protein, hemoglobin (Hb), onto the surface of glassy carbon electrode (GCE). The multiporous nanofiber of SnO₂ that has very high surface area is synthesized by using electrospinning technique through controlling the tin precursor concentration. Since the constructed MPNFs of SnO₂ exposes very high surface area, it increases the efficiency for biomolecule-loading. The morphology of fabricated electrodes is examined by SEM observation and the absorbance spectra of Hb/(MPNFs) of SnO₂ are studied by UV-Vis analysis. Cyclic Voltammetry and amperometry are employed to study and optimize the performance of the resulting fabricated electrode. After fabrication of the electrode with the Hb and MPNFs of SnO₂, a direct electron transfer between the protein's redox centre and the glassy carbon electrode was established. The modified electrode has showed a couple of redox peak located at -0.29 V and -0.18 V and found to be sensitive to H₂O₂. The fabricated electrode also exhibited an excellent electrocatalytic activity towards the reduction of H₂O₂. The catalysis currents increased linearly to the H₂O₂ concentration in a wide range of 5.0×10-6-1.5×10-4 M. Overall experimental results show that MPNFs of SnO₂ has a role towards the enhancement of the electroactivity of Hb at the electrode surface. Thus the MPNFs of SnO₂ is a very promising candidate for future biosensor applications.

Matched MeSH terms: Immobilized Proteins*
Investigating membrane morphology and quantity of immobilized protein for the development of lateral flow immunoassay

Ahmad AL, Low SC, Shukor SR, Ismail A

J Immunoassay Immunochem, 2012 Jan;33(1):48-58.
PMID: 22181820 DOI: 10.1080/15321819.2011.591479

This study was aimed at gaining a quantitative understanding of the effect of protein quantity and membrane pore structure on protein immobilization. The concentration of immobilized protein was measured by staining with Ponceau S and measuring its color intensity. In this study, both membrane morphology and the quantity of deposited protein significantly influenced the quantity of protein immobilization on the membrane surface. The sharpness and intensity of the red protein spots varied depending on the membrane pore structure, indicating a dependence of protein immobilization on this factor. Membranes with smaller pores resulted in a higher color density, corresponding to enhanced protein immobilization and an increased assay sensitivity level. An increased of immobilized volume has a significant jagged outline on the protein spot but, conversely, no difference in binding capacity.

Matched MeSH terms: Immobilized Proteins/analysis*; Immobilized Proteins/immunology; Immobilized Proteins/chemistry
Comprehensive spectroscopic studies of synergism between Gadong starch based carbon dots and bovine serum albumin

Sonthanasamy RSA, Sulaiman NMN, Tan LL, Lazim AM

Spectrochim Acta A Mol Biomol Spectrosc, 2019 Jul 05;218:85-96.
PMID: 30954801 DOI: 10.1016/j.saa.2019.03.108

Carbon dots (C-dots) were used to study the binding mechanisms with serum protein, bovine serum albumin (BSA) by using two notable binding systems known as non-covalent and covalent interaction. Interaction between C-dots and BSA were estimated by Stern-Volmer equation and Double Log Regression Model (DLRM). According to the fluorescent intensity, quenching of model carrier protein by C-dots was due to dynamic quenching for non-covalent and static quenching for covalent binding. The binding site constant, KA and number of binding site, for covalent interaction is 1754.7L/mol and n≈1 (0.6922) were determined by DLRM on fluorescence quenching results. The blue shift of the fluorescence spectrum, from 450nm to 421nm (non-covalent) and 430nm (covalent) and suggested that both the microenvironment of C-dots and protein changed in relation to the protein concentration. The fluorescence intensity results show that protein structure has a significant role in Protein-C-dots interactions and type of binding influence physicochemical properties of C-dots differently. Understanding to this bio interface is important to utilize both quantum dots and biomolecules for biomedical field. It can be a useful guideline to design further applications in biomedical and bioimaging.

Matched MeSH terms: Immobilized Proteins/metabolism; Immobilized Proteins/chemistry
Fulltext Gold nanoparticle-based colorimetric method for the detection of prostate-specific antigen

Xia N, Deng D, Wang Y, Fang C, Li SJ

Int J Nanomedicine, 2018;13:2521-2530.
PMID: 29731627 DOI: 10.2147/IJN.S154046

Background: Prostate-specific antigen (PSA), a serine protease, is a biomarker for preoperative diagnosis and screening of prostate cancer and monitoring of its posttreatment.
Methods: In this work, we reported a colorimetric method for clinical detection of PSA using gold nanoparticles (AuNPs) as the reporters. The method is based on ascorbic acid (AA)-induced in situ formation of AuNPs and Cu2+-catalyzed oxidation of AA. Specifically, HAuCl4 can be reduced into AuNPs by AA; Cu2+ ion can catalyze the oxidation of AA by O2 to inhibit the formation of AuNPs. In the presence of the PSA-specific peptide (DAHSSKLQLAPP)-modified gold-coated magnetic microbeads (MMBs; denoted as DAHSSKLQLAPP-MMBs), complexation of Cu2+ by the MMBs through the DAH-Cu2+ interaction depressed the catalyzed oxidation of AA and thus allowed for the formation of red AuNPs. However, once the peptide immobilized on the MMB surface was cleaved by PSA, the DAHSSKLQ segment would be released. The resultant LAPP fragment remaining on the MMB surface could not sequestrate Cu2+ to depress its catalytic activity toward AA oxidation. Consequently, no or less AuNPs were generated.
Results: The linear range for PSA detection was found to be 0~0.8 ng/mL with a detection limit of 0.02 ng/mL. Because of the separation of cleavage step and measurement step, the interference of matrix components in biological samples was avoided.
Conclusion: The high extinction coefficient of AuNPs facilitates the colorimetric analysis of PSA in serum samples. This work is helpful for designing of other protease biosensors by matching specific peptide substrates.

Matched MeSH terms: Immobilized Proteins/metabolism; Immobilized Proteins/chemistry
Magnetic bead-based semi-automated phage display panning strategy for the directed evolution of antibodies

Ch'ng ACW, Konthur Z, Lim TS

Methods Enzymol, 2020;630:159-178.
PMID: 31931984 DOI: 10.1016/bs.mie.2019.10.023

Directed evolution is a proven approach to fine tune or modify biomolecules for various applications ranging from research to industry. The process of evolution requires methods that are capable of not only generating genetic diversity but also to distinguish the variants of desired characteristics. One method that is synonymous with directed evolution of proteins is phage display. Here, we present a protocol describing the application of magnetic nanoparticles coupled with a processor to carry out the identification of monoclonal antibodies (mAbs) from a diverse antibody library via phage display. Target antigens are coupled to magnetic nanoparticles as the solid phase for the isolation of the binding mAbs via affinity. A gradual enrichment in clones would result in increasing ELISA readouts with increasing rounds of panning. During monoclonal level analysis, positivity can be deduced with comparison to background and controls. The biopanning process can also be adopted for the directed evolution of enzymes, scaffold proteins or even peptides.

Matched MeSH terms: Immobilized Proteins/immunology; Immobilized Proteins/chemistry
Inclusion bodies of recombinant Epstein-Barr virus capsid antigen p18 as potential immobilized antigens in enzyme immunoassays for detection of nasopharyngeal carcinoma

Lim CS, Goh SL, Kariapper L, Krishnan G, Lim YY, Ng CC

Clin Chim Acta, 2015 Aug 25;448:206-10.
PMID: 26164385 DOI: 10.1016/j.cca.2015.07.008

Development of indirect enzyme-linked immunosorbent assays (ELISAs) often utilizes synthetic peptides or recombinant proteins from Escherichia coli as immobilized antigens. Because inclusion bodies (IBs) formed during recombinant protein expression in E. coli are commonly thought as misfolded aggregates, only refolded proteins from IBs are used to develop new or in-house diagnostic assays. However, the promising utilities of IBs as nanomaterials and immobilized enzymes as shown in recent studies have led us to explore the potential use of IBs of recombinant Epstein-Barr virus viral capsid antigen p18 (VCA p18) as immobilized antigens in ELISAs for serologic detection of nasopharyngeal carcinoma (NPC).

Matched MeSH terms: Immobilized Proteins/immunology*; Immobilized Proteins/chemistry
Wide dynamic range of surface-plasmon-resonance-based assay for hepatitis B surface antigen antibody optimal detection in comparison with ELISA

Tam YJ, Zeenathul NA, Rezaei MA, Mustafa NH, Azmi MLM, Bahaman AR, et al.

Biotechnol Appl Biochem, 2017 Sep;64(5):735-744.
PMID: 27506960 DOI: 10.1002/bab.1528

Limit of detection (LOD), limit of quantification, and the dynamic range of detection of hepatitis B surface antigen antibody (anti-HBs) using a surface plasmon resonance (SPR) chip-based approach with Pichia pastoris-derived recombinant hepatitis B surface antigen (HBsAg) as recognition element were established through the scouting for optimal conditions for the improvement of immobilization efficiency and in the use of optimal regeneration buffer. Recombinant HBsAg was immobilized onto the sensor surface of a CM5 chip at a concentration of 150 mg/L in sodium acetate buffer at pH 4 with added 0.6% Triton X-100. A regeneration solution of 20 mM HCl was optimally found to effectively unbind analytes from the ligand, thus allowing for multiple screening cycles. A dynamic range of detection of ∼0.00098-0.25 mg/L was obtained, and a sevenfold higher LOD, as well as a twofold increase in coefficient of variance of the replicated results, was shown as compared with enzyme-linked immunosorbent assay (ELISA). Evaluation of the assay for specificity showed no cross-reactivity with other antibodies tested. The ability of SPR chip-based assay and ELISA to detect anti-HBs in human serum was comparable, indicating that the SPR chip-based assay with its multiple screening capacity has greater advantage over ELISA.

Matched MeSH terms: Immobilized Proteins/metabolism*; Immobilized Proteins/chemistry
Process evaluation and in vitro selectivity analysis of aptamer-drug polymeric formulation for targeted pharmaceutical delivery

Tan KX, Lau SY, Danquah MK

Biomed Pharmacother, 2018 May;101:996-1002.
PMID: 29635910 DOI: 10.1016/j.biopha.2018.03.052

Targeted drug delivery is a promising strategy to promote effective delivery of conventional and emerging pharmaceuticals. The emergence of aptamers as superior targeting ligands to direct active drug molecules specifically to desired malignant cells has created new opportunities to enhance disease therapies. The application of biodegradable polymers as delivery carriers to develop aptamer-navigated drug delivery system is a promising approach to effectively deliver desired drug dosages to target cells. This study reports the development of a layer-by-layer aptamer-mediated drug delivery system (DPAP) via a w/o/w double emulsion technique homogenized by ultrasonication or magnetic stirring. Experimental results showed no significant differences in the biophysical characteristics of DPAP nanoparticles generated using the two homogenization techniques. The DPAP formulation demonstrated a strong targeting performance and selectivity towards its target receptor molecules in the presence of non-targets. The DPAP formulation demonstrated a controlled and sustained drug release profile under the conditions of pH 7 and temperature 37 °C. Also, the drug release rate of DPAP formulation was successfully accelerated under an endosomal acidic condition of ∼pH 5.5, indicating the potential to enhance drug delivery within the endosomal micro-environment. The findings from this work are useful to understanding polymer-aptamer-drug relationship and their impact on developing effective targeted delivery systems.

Matched MeSH terms: Immobilized Proteins/pharmacology