QCM Temelli Homosistein Nanobiyosensörünün Gerçek Zamanlı Tayin Sisteminde Optimizasyonu

Öz

Metiyonin amino asitinden türeyen bir amino asit olan homosistein, damar hastalıkları için önemli bir risk faktörü olduğundan hızlı ve ucuz bir yöntemle tayini son yıllarda üzerinde çalışılan ve gittikçe artan öneme sahip bir araştırma konusudur. Sunulan bu araştırma kapsamında, Kuartz Kristal mikrobalans (QCM) yöntemi kullanılarak homosisteinin gerçek zamanlı tayini yapılmıştır. QCM tekniği özellikle nano düzeylerde bazı maddelerin varlığının nicel ve/veya nitel olarak tespitinde kullanılmaktadır. Kristalin elektrodunun yüzeyine özgül olarak immobilize edilen tanıyıcı maddenin hedef maddeyi yakaladığı mikrogravimetrik tayin prensibine dayanmaktadır. Yapılan çalışmada, kuartz kristal mikroterazi (QCM) tekniği ile homosisteinin sıvı ortamda ve sürekli akış sisteminde gerçek zamanlı bir yöntemle tayini mevcut yöntemlere göre daha hızlı olarak gerçekleştirilmiştir. Bu amaçla çalışmanın ilk aşamasında altın (Au) elektrotlu kristal yüzeylerinin modifikasyonu gerçekleştirilmiştir. Yüzey yıkama ve aktivasyon işleminden sonra yüzeye sisteamin ve daha bifonksiyonel bir yapıda olan glutaraldehid (GA) bağlanması gerçekleştirilmiştir. GA bağlanmış yüzeylere homosisteini özgül olarak tanıyan biyolojik ligand Bovin Serum Albumin (BSA) immobilize edilmiştir. En uygun BSA derişimi olarak 0,1 mg/ml seçilmiştir. Bu BSA yüzey derişimi ile yapılan homosistein bağlanma deneyleri ile 0,01-0,5 μM aralığında homosistein kalibrasyonu gerçekleştirilmiş ve en düşük tayin edilebilir değer olarak 0,01 μM yani 10 nM düzeyinde homosistein değeri belirlenmiştir. Böylece bu önerilen sistemle nanodüzeyde homosistein tayini yapılabilmiştir. Çalışmanın ikinci aşamasında gerçek zamanlı (real-time) homosistein ölçümleri ile yöntemin verimliliği, tekrarlanabilirliği ve tayin edilebilecek en düşük homosistein değerinin tespit edilmesi sağlanmıştır. Sonuçta damar hastalıklarının önemli bir belirteci olan homosisteinin tayininde geliştirilen sistemin yeni bir yöntem olarak kullanım potansiyeli olduğu düşünülmektedir.

Anahtar Kelimeler

• Kuartz kristal mikroterazi (QCM)
• Homosistein
• Yüzey modifikasyonu
• Gerçek zamanlı tayin
• Optimizasyon

Optimization of QCM-Based Homocysteine Nanobiosensor in Real-Time Detection System

Abstract

Homocysteine, which is a methionine amino acid derived amino acid is an important risk factor for vascular deseases so, its dedection by a rapid and cheap method is under investigation and is an research theme with gradually increasing importance. In the scope of the presented study, the real time detection of homocysteine by using Quartz Crystal Microbalance (QCM) method was performed. QCM technique is used for quantitative and/or qualitative determination of some materials especially which are present in nanoscale levels. The analysis lies on the microgravimetric analysis where the specific compound attached to the surface of crystal capture the target compound. The detection of homocysteine with QCM technique in liquid and continuous flow system was achieved in a real time method faster than other available methods. For the purpose, the modification of gold (Au) crystal surfaces was performed in the first part of the study. Cystamine was immobilized after surface washing and activation process and then binding of bifunctional glutaraldehyde (GA) was achieved. Homocysteine specific recognizing biological ligand Bovine Serum Albumin (BSA) was immobilized to glutaraldehyde coupled surfaces. The most convinient BSA concentration was selected as 0,1 mg/ml. and homocysteine coupling values was determined and the calibration of homocystein was achieved. Homocysteine calibration curve was realized by homocysteine coupling experiments with this BSA surface concentration and the minimum detection limit was found as 0,01 µM which coincide to 10 nM homocysteine value. Hence, homocysteine was detected in nano level in the proposed system. In the second part of the research, productivity, reproducibility, and minimum detection limit of the method was determined by the experiments of real-time detection of homocystein. Thus, it is thought that the system developed for the determination of homocysteine, which is an important marker of vascular diseases, has the potential to be used as a new method.

Keywords

• Quartz crystal microbalance (QCM)
• Homocysteine
• Surface modification
• Real-time detection
• Optimization

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