Homosistein tayini amaçlı QCM-temelli biyosensör

Öz

Yüksek plazma homosistein düzeyleri, kardiyovasküler, serebrovasküler ve peripheral arterial hastalık risklerini arttırabilmektedir. Bunun yanında doğumsal bozukluklar gibi diğer çeşitli patolojiler, Alzheimer hastalığına ve diğer gerilikler, osteoporoz, şeker ve böbrek hastalıkları da yüksek homosistein değerlerinin sonucu olabilmektedir. Sunulan araştırma çalışmasının amacı Kuartz Kristal Mikroterazi (QCM) biosensörü kullanarak Homosisteinin (Hsis) tayinini gerçekleştirmektir. Sıcaklık kontrollü QCM sistemi, yerli olarak tasarlanmış ve üretilmiş bir cihaz olup gümüş elektrodlu kuartz kristal kullanılmıştır. Gümüş elektrod kristal yüzeylerinin modifikasyonu sodium hidroksit, aseton ve methanolün ardışık olarak uygulanarak yapılan yüzey yıkama işlemi ile sağlanmıştır. Daha sonra sisteamin ile kendiliğinden tek tabaka oluşumu ile uzatma kolu/ligand oluşumu gerçekleştirilmesi amacıyla tek tabakanın serbest ucuna glutaraldehitin kimyasal olarak bağlanması sağlanarak yeni bir fonksiyonel yüzey elde edilmiştir. Glutaraldehit bağlı yüzeylere homosisteine özgü ligand olan anti-Homosistein antikoru immobilize edilmiştir. Her modifikasyon basamağı için frekans değerlerinin değişimi ölçülmüştür. Antikor çözeltisinin seyrelme oranı modifiye yüzeyler kullanılarak optimize edilmiştir. En düşük antikor derişimi olan 1/10000 v/v oranı optimize antikor oranı olarak belirlenmiştir. Homosisteinin en düşük tayin sınırı 0.1 µM olarak tespit edilmiştir ve kalibrasyon eğrilerinin doğrusal aralıkları 0.1-2.0 µM ve 10-50 µM olarak bulunmuştur. Belirtilen doğrusal aralıklarda Hsis değerleri oldukça yüksek doğrusallık (sırası ile R2=0.9813 and 0.9875) göstermiştir. Bağıl standart sapmanın duyarlılığı % 10’dan küçük hesaplanmıştır. Sonuç olarak homosistein tayininin hem nano hem mikro molar derişim değerlerinde yapılabileceği bulunmuştur. Ek olarak tasarlanan biyosensör istenen kararlılık ve tekrarlanabilirlik göstermiştir. En son olarak Homosistein analizinde kullanıma yönelik yeni bir yöntem önerilerek geliştirilmiş ve bu hızlı, ucuz ve daha az ön işlemi olan QCM tekniği ile homosistein tayini başarı ile yapılabilmektedir.

Anahtar Kelimeler

• Homosistein
• Anti-Homosistein antikor
• Kuartz Kristal Mikrodenge (QCM)
• Yüzey modifikasyonu

QCM-based biosensor for the Detection of Homocysteine

Öz

High plasma homocysteine levels can cause an increased risk of cardiovascular, cerebrovascular, and peripheral arterial diseases. Besides this, Alzheimer’s disease and other dementias, osteoporosis, diabetes and renal disease due to folate and B-vitamin deficiency, various drugs or pre-existing atherosclerotic diseases may be the result of high homocysteine levels. The presented research work aimedto perform the detection of Homocysteine (Hcy) by using Quartz Crystal Microbalance (QCM) biosensor. The temperature controlled QCM system was a home-made designed and constructed equipment which can use silver electrod quartz crystal. The modification of silver electrod quartz crystals surfaces was achieved by the surface cleaning process with sodium hydroxide, acetone and methanol in a consecutive manner. Then self-assembled monolayer of cysteamine and chemical coupling of glutaraldehyde (GA) to free end of monolayer was achieved to create the new functional surface in order to complete the formation of spacer arm/ligand. Homocysteine specific recognizing ligand, anti-Homocysteine antibody was immobilized to glutaraldehyde coupled surfaces. The change in resonance frequency values were measured for each modification step. The optimization of dilution ratio of the antibody solution was performed to modified surfaces. The least dilution ratio of antibody, 1/10000 v/v, was determined as optimum antibody ratio. The detection of homocystein was analysed at a detection limit of 0.1 µM and the linear ranges of calibration curves were estimated as 0.1-2.0 µM and 10-50 µM. Homocysteine values indicated good linearities (R2=0.9813 and 0.9875, respectively). The relative standart deviation (RSD %) for precision was calculated as less than 10%. In conclusion, it was found that the detection of homocysteine can be done both in nano- and micro-molar concentration levels. Additionally, designed biosensor showed desired stability and reproducibility. Finally, a new method different from the present methods for the use in the analysis of Hcy was proposed and developed which detects homocysteine by designed QCM technique with a rapid, cheeper and less pretreatment processes. Additionally, homocysteine detection was performed in nano- and micro- molar concentration values.

Anahtar Kelimeler

• Homocysteine
• anti-Homocysteine antibody
• Quartz Crystal Microbalance (QCM)
• Surface modifikation

Kaynakça

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