USABILITY OF PROTEIN-INORGANIC HYBRID CONJUGATE IN ELISA SYSTEM

Yıl 2020, Cilt: 9 Sayı: 2, 226 - 238, 30.07.2020

Murat Ekremoğlu , Cevahir Altınkaynak

https://doi.org/10.18036/estubtdc.604509

Cited By: 1

Öz

In the Enzyme-linked immunosorbent assay method (ELISA), it
is important to increase the immobilization efficiency of the functional
molecules on the microplate and increase the signal-to-noise ratio in order to
detect the target molecules with high sensitivity and selectivity. For this
purpose, antibody functionalized materials can generate remarkable signal
amplification with high enzyme capacity by using conventional immobilization
methods. Recently, hybrid structures containing protein/enzyme and Cu₃(PO₄)₂,
which is a different enzyme immobilization method, have higher catalytic
activity compared to the free form of protein-containing molecules as they have
hierarchical microstructures and form large active surface areas.

In this study; Using protein-inorganic hybrid structure
synthesis method, hybrid functionalized conjugate systems with enzyme, antibody
and Cu₃(PO₄)₂ were synthesized all together
and characterization of the resulting structures was performed by SEM, EDX, XRD
and FTIR analysis. When the findings obtained from the researches were
evaluated, the hybrid nano structure showed 183.5 EU/mg peroxidase activity and
free HRP enzyme showed 59.01 EU/mg activity. The performance of the ELISA
system was measured using hybrid conjugate constructs prepared using various
TNF-alpha specific antibodies at a concentration of 5-1000 mg mL^-1. The performance of hybrid
conjugate structure containing multiple organic molecules in ELISA system was
higher than other structures. This method is a highly practical method that can
replace enzyme-labeled antibody method in ELISA.

Anahtar Kelimeler

Hybrid conjugate, Protein-inorganic hybrid structure, ELISA

ELISA SİSTEMİNDE PROTEİN-İNORGANİK HİBRİT KONJUGATIN KULLANILABİLİRLİĞİ

Öz

Enzim bağlı immünosorbent analizinde (ELISA); hedef
molekülleri yüksek hassasiyet ve seçicilikle tespit etmek için fonksiyonel
moleküllerin mikro plaka üzerindeki immobilizasyon verimlerinin arttırılması ve
sinyal-gürültü oranının yükseltilmesi önemlidir.  Bu amaçla konvansiyonel immobilizasyon
yöntemleri kullanılarak antikorla fonksiyonelleştirilmiş malzemeler yüksek
enzim kapasitesi ile dikkate değer sinyal artışına neden olabilir) Son yıllarda
farklı bir enzim immobilizasyon yöntemi olan protein/enzim ve Cu₃(P0₄)₂
içeren hibrit yapılar hiyerarşik mikro yapılara sahip olmaları ve geniş aktif
yüzey alanları oluşturmaları neticesinde içeriğindeki protein yapılı moleküllerin
serbest formuna göre daha yüksek katalitik aktivite gösterebilmektedir.

Bu çalışma kapsamında; protein-inorganik hibrit yapı sentez metodu
kullanılarak enzimi, antikoru ve Cu₃(P0₄)₂’ın
hepsi bir arada olacak şekilde antikorla fonksiyonelleştirilmiş hibrit konjugat
sistemleri sentezlenmiş, oluşan yapıların karakterizasyonları SEM, EDX, XRD ve FTIR
analizleri ile gerçekleştirilmiştir. Araştırmalar sonucu elde edilen bulgular
değerlendirildiğinde, hibrit nano yapı 183,5 EU/mg peroksidaz aktivitesi
gösterirken serbest HRP enzimi 59,01 EU/mg aktivite göstermiştir. TNF-alfa’ya
spesifik çeşitli antikorlar kullanılarak hazırlanan hibrit konjugat yapılar 5-1000 µg mL^-1
aralığındaki konsantrasyonlarda kullanılarak ELISA sistemindeki
performansı ölçülmüştür. Çoklu organik molekül içeren hibrit konjugat
yapının ELISA sistemindeki performansı diğer yapılara göre daha yüksek
bulunmuştur. Bu metot; ELISA’da antikorların enzimle işaretlenmesi yöntemi yerine
geçebilecek pratik olarak uygulanabilirliği kanıtlanmış bir uygulamadır.

Anahtar Kelimeler

Hibrit konjugat, protein-inorganik hibrit yapı, ELISA

Kaynakça

• 1.Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G. Immunochemistry 8, 871–874 (1971).
• 2.Wei T, Du D, Zhu M.-J, Lin Y, Dai Z. An Improved Ultrasensitive Enzyme-Linked Immunosorbent Assay Using Hydrangea-Like Antibody-Enzyme-Inorganic Three-in-One Nanocomposites. ACS Appl. Mater. Interfaces 8, 6329–6335 (2016).
• 3.Ram J, Nakane P, Rawlinson D, Pierce G. Enzyme labelled antibodies for ultrastructural studies. Fed Proc 25, (1966).
• 4.Shah K, Maghsoudlou P. Enzyme-linked immunosorbent assay (ELISA): the basics. Br. J. Hosp. Med. Lond. Engl. 2005 77, C98-101 (2016).
• 5.Ball JM, Henry NL, Montelaro RC, Newman MJA. versatile synthetic peptide-based ELISA for identifying antibody epitopes. J. Immunol. Methods 171, 37–44 (1994).
• 6.Stearns NA, Zhou S, Petri M, Binder SR, Pisetsky DS. The Use of Poly-L-Lysine as a Capture Agent to Enhance the Detection of Antinuclear Antibodies by ELISA. PLOS ONE 11, e0161818 (2016).
• 7.Tong S, Ren B, Zheng Z, Shen H, BaoG , Tiny Grains Give Huge Gains: Nanocrystal-Based Signal Amplification for Biomolecule Detection - ACS Nano (ACS Publications). Available at: https://pubs.acs.org/doi/10.1021/nn400733t. (Accessed: 11th May 2019)
• 8.Jeanson A, Cloes JM, Bouchet M, Rentier B. Comparison of conjugation procedures for the preparation of monoclonal antibody-enzyme conjugates. J. Immunol. Methods 111, 261–270 (1988).
• 9.Speroni F, Elviri L, Careri M, Mangia A. Magnetic particles functionalized with PAMAM-dendrimers and antibodies: a new system for an ELISA method able to detect Ara h3/4 peanut allergen in foods. Anal. Bioanal. Chem. 397, 3035–3042 (2010).
• 10.Lin H. et al. Modified Enzyme-Linked Immunosorbent Assay Strategy Using Graphene Oxide Sheets and Gold Nanoparticles Functionalized with Different Antibody Types - Analytical Chemistry (ACS Publications). Available at: https://pubs.acs.org/doi/10.1021/ac401075u. (Accessed: 11th May 2019)
• 11.Qu Z. et al. Ultrasensitive ELISA Using Enzyme-Loaded Nanospherical Brushes as Labels. Anal. Chem. 86, 9367–9371 (2014).
• 12.Ge J, Lei J, Zare RN. Protein-inorganic hybrid nanoflowers. Nat. Nanotechnol. 7, 428–432 (2012).
• 13.Altinkaynak C, Kocazorbaz E, Özdemir N, Zihnioglu F. Egg white hybrid nanoflower (EW-hNF) with biomimetic polyphenol oxidase reactivity: Synthesis, characterization and potential use in decolorization of synthetic dyes. Int. J. Biol. Macromol. 109, 205–211 (2018).
• 14.Shende P, Kasture P, Gaud RS. Nanoflowers: the future trend of nanotechnology for multi-applications. Artif. Cells Nanomedicine Biotechnol. 46, 413–422 (2018).
• 15.Lee SW, Cheon SA, Kim MI, Park TJ. Organic-inorganic hybrid nanoflowers: types, characteristics, and future prospects. J. Nanobiotechnology 13, 54 (2015).
• 16.Altinkaynak C, Tavlasoglu S, Özdemir N, Ocsoy I. A new generation approach in enzyme immobilization: Organic-inorganic hybrid nanoflowers with enhanced catalytic activity and stability. Enzyme Microb. Technol. 93–94, 105–112 (2016).
• 17.Altinkaynak C. et al. Preparation of lactoperoxidase incorporated hybrid nanoflower and its excellent activity and stability. Int. J. Biol. Macromol. 84, 402–409 (2016).
• 18.Somturk B. et al. Synthesis of urease hybrid nanoflowers and their enhanced catalytic properties. Enzyme Microb. Technol. 86, 134–142 (2016).
• 19.Lin Z. et al. Facile synthesis of enzyme-inorganic hybrid nanoflowers and its application as a colorimetric platform for visual detection of hydrogen peroxide and phenol. ACS Appl. Mater. Interfaces 6, 10775–10782 (2014).
• 20.Sun J. et al. Multi-enzyme co-embedded organic-inorganic hybrid nanoflowers: synthesis and application as a colorimetric sensor. Nanoscale 6, 255–262 (2014).
• 21.Wu Z. et al. Enantioselective transesterification of (R,S)-2-pentanol catalyzed by a new flower-like nanobioreactor. RSC Adv. 4, 33998–34002 (2014).
• 22.Zhang Y, Ge J, Liu Z. Enhanced Activity of Immobilized or Chemically Modified Enzymes. ACS Catal. 5, 4503–4513 (2015).
• 23.Yin Y. et al. An enzyme–inorganic hybrid nanoflower based immobilized enzyme reactor with enhanced enzymatic activity. J. Mater. Chem. B 3, 2295–2300 (2015).
• 24.Gulmez C, Altinkaynak C, Özdemir N, Atakisi O. Proteinase K hybrid nanoflowers (P-hNFs) as a novel nanobiocatalytic detergent additive. Int. J. Biol. Macromol. 119, 803–810 (2018).
• 25.Zhu L. et al. Rapid Detection of Phenol Using a Membrane Containing Laccase Nanoflowers. Chem. – Asian J. 8, 2358–2360 (2013).
• 26.Zeng J, Xia Y. Hybrid nanomaterials: Not just a pretty flower. Nat. Nanotechnol. 7, 415–416 (2012).
• 27.Lei Z. et al. Recent advances in biomolecule immobilization based on self-assembly: organic–inorganic hybrid nanoflowers and metal–organic frameworks as novel substrates. J. Mater. Chem. B 6, 1581–1594 (2018).
• 28.Wang R, Zhang Y, Lu D, Ge J, Liu Z, Zare RN. Functional protein–organic/inorganic hybrid nanomaterials. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology - Wiley Online Library. Available at: https://onlinelibrary.wiley.com/doi/full/10.1002/wnan.1210. (Accessed: 21st May 2019)