Next-generation biosensor systems for environmental water quality monitoring
Çevresel su kalitesi izleme için yeni nesil biyoensör sistemleri
- Tez No: 889980
- Danışmanlar: PROF. DR. APRİL Z. GU
- Tez Türü: Doktora
- Konular: Biyomühendislik, Bioengineering
- Anahtar Kelimeler: Biyosensörler, Biyoteknoloji, Biosensors, Biotechnology
- Yıl: 2015
- Dil: İngilizce
- Üniversite: Northeastern Unıversıty
- Enstitü: Yurtdışı Enstitü
- Ana Bilim Dalı: Biyomühendislik Ana Bilim Dalı
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: Belirtilmemiş.
Özet
Suyumuzda artan sayıda yeni ortaya çıkan kirleticilerle ilgili tanınan ve bilinmeyen sağlık riskleri ile zararlı çevresel etkiler, hepimiz için ciddi bir tehdit oluşturmaktadır. Bu durum, bu kirleticilerin akıbetini ve taşınmasını anlamak, maruziyet değerlendirmesi yapmak ve bu tehditleri ortadan kaldırmak için düzenleyici karar alma süreçlerinde gerekli olan duyarlı, uygulanabilir ve etkili su kirleticileri izleme yöntemlerine yönelik acil bir ihtiyaç doğuruyor. Mevcut su kirleticilerinin tespiti çoğunlukla zaman alıcı, pahalı ve yüksek eğitimli personel gerektiren kimyasal analizlere (örneğin, GC-MS, HPLC-MS, vb.) dayanmaktadır ve bu nedenle çevredeki kirleticilerin yeterince sık izlenmesine olanak tanımaz. Daha kapsamlı, yerinde izleme sağlamak için duyarlı, basit, hızlı, maliyet etkin ve taşınabilir tespit yöntemlerine büyük bir ihtiyaç vardır. Biyosensörler gibi algılama teknolojilerinin, çevresel izleme için uygun bir alternatif veya tamamlayıcı analitik yöntemler olarak hizmet ettiği gösterilmiştir. Genel olarak, biyosensörler, biyolojik bir malzeme (örneğin, enzimler, antikorlar, nükleik asitler, doğal ürünler vb.) veya bir biyomimik (örneğin, sentetik reseptörler, baskılanmış polimerler vb.) içeren ve bir fizikokimyasal dönüştürücü veya mikro sistemle bütünleşmiş analitik algılama cihazları olarak tanımlanır. Bu dönüştürücü, optik, elektrokimyasal, termometrik, piezoelektrik, manyetik veya mikromekanik olabilir. Çevresel kirleticilerin tespitinde biyosensörlerin geleneksel analitik tekniklere göre başlıca avantajları; taşınabilirlik, minyatürleştirilebilme, yerinde çalışma olanağı, daha kolay kullanım, daha düşük maliyet ve kirleticilerin kompleks matrislerde minimum örnek hazırlığı ile ölçülebilmesidir. Bu çalışmanın amaçları, çeşitli çevresel kirleticilerin hızlı, yeniden kullanılabilir, kolay kullanımlı, spesifik, gerçek zamanlı ve yerinde tespiti için geliştirilen ve potansiyel olarak taşınabilir biyosensör sistemlerinin geliştirilmesi ve uygulanmasının gösterilmesidir. İki ayrı algılama sistemi kullanarak bir dizi biyosensör geliştirdik; biri sönümlenen dalga optik fiber sistemi ve diğeri SWCNT tabanlı nanobiyosensör sistemi. Her bir algılama sistemi için, antikor gibi geleneksel biyotanıma elemanları ve aptamer, DNAzim ve G-kuadrupleks gibi yeni nesil biyotanıma elemanlarının kullanımını araştırdık. Bu iki algılama sistemi ve çeşitli biyotanıma elemanları ile farklı sınıflardaki çevresel kirleticiler için biyosensörler geliştirebileceğimizi gösterdik. Bu kirleticiler arasında metaller, organik kirleticiler (EDC'ler), antibiyotikler, virüsler ve bakteriler bulunmaktadır. İlk olarak, Kurşun iyonu (Pb2+), 17-β Estradiol, Bisfenol A, E coli 0157H:7 ve adenovirüsün gerçek zamanlı tespiti için aptamerler, DNAzim ve antikor gibi biyotanıma elemanlarını kullanarak taşınabilir bir optik fiber algılama sistemi üzerinde beş biyosensör geliştirdik. Optik fiber sistemlerinin verimliliği, doğruluğu, düşük maliyeti ve yüksek matris etkili numunelerde hızlı tespit için uygunluğu nedeniyle, geleneksel algılama yöntemlerine umut verici alternatiflerdir.
Özet (Çeviri)
The recognized and unknown health risks and the harmful environmental impacts associated with the ever-increasing number of emerging pollutants in our water present a serious threat to us all. This poses a pressing need for sensitive, feasible and effective water pollutants monitoring methods, which are required for understanding the fate and transport of these pollutants, for exposure assessment and for regulatory decision making to eliminate these threats. Current water pollutants detection mostly rely on chemical analysis (i.e., GC-MS, HPLC-MS, etc.) that are often time consuming, expensive and require highly trained personnel and therefore do not allow sufficiently frequent monitoring of contaminants in the environment. There is a great need for sensitive, simple, rapid, cost-effective and portable detection methods to allow more comprehensive, on-site monitoring with special and temporal resolutions. Sensing technologies such as biosensors have been shown to serve as a suitable alternative or as complementary analytical methods for environmental monitoring. In general, biosensors are defined as analytical sensing devices incorporating a biological material (i.e., enzymes, antibodies, nucleic acids, natural products, etc.), or a biomimic (i.e., synthetic receptors, imprinted polymers, etc.) intimately integrated within a physicochemical transducer or transducing microsystem, which may be optical, electrochemical, thermometric, piezoelectric, magnetic or micromechanical. The main advantages of biosensors, over traditional analytical techniques for the detection of environmental contaminants, are the possibility of portability, miniaturization and work on-site, easier to use, less costly and the ability to measure pollutants in complex matrices with minimal sample preparation. The objectives of this study are to develop and demonstrate the application of improved potentially portable biosensor systems for rapid, reusable, easy-to-use, specific, real time and on-site detection for various environmental pollutants with either enhanced performance or novel sensing mechanisms. We have developed a number of biosensors using two separate sensing systems; one is evanescent wave optical fiber system and another is SWCNT based nanobiosensor system. For each of the sensing systems, we explored the employment of various biorecognition elements including conventional element such as antibody and newer generation biorecognition elements such as aptamer, DNAzyme and G-quadruplex. We have demonstrated that with these two sensing systems and with various biorecognitions comments, we can develop biosensors for different classes of environmental pollutants including metals, organic pollutants (EDCs), antibiotics, virus and bacteria. iii First, we developed five biosensors on a portable optical fiber sensing system for real-time detection of Lead ion (Pb2+), 17-β Estradiol, Bisphenol A, E coli 0157H:7 and adenovirus, using aptamers, DNAzyme and antibody as biorecognition elements, respectively. Because of the optical fiber systems' efficiency, accuracy, low cost, and suitability for rapid detection in high matrix effect samples, they are promising alternatives to traditional sensing methods. Additionally, with these properties, the optical fiber based biosensor systems are quite appropriate for real-time, on-site and multi detections. Lead ion (Pb2+) is one of the most toxic metallic pollutants that can cause neurological, reproductive, cardiovascular, and developmental disorders even at very low levels (<100μg/L in blood). We developed a DNAzyme based portable, inexpensive, and easy-to-use evanescent wave fiber-optic biosensor for the detection of Pb2+ in aqueous solution with the detection limit of 1.03 nM and detection time of 15 minutes which are better or comparable to previous biosensors and comparable to chemical analytical. Two other optical fiber biosensors were developed for detection of two contaminants of emerging concerns (CECs), namely 17-β Estradiol and Bisphenol A (BPA). These two CECs are widely spread in water and they have effects on human and other organisms' reproductive systems and cause growing concerns. Thus, simple, selective, and sensitive detection methods for these contaminants are in urgent need. With the newly developed system for both CECs, we observed sensitive detection limit (0.5 to 1.5 ng/ml) with less than 30 minutes detection time lengths, which are comparable with the past works. Additionally, the developed systems have superiority over other analytical systems with respect to stability and regeneration capability. It is known that, E.coli is a good indicator for potential enteric pathogens in waters. Thus, a rapid and simple detection method for E.coli is extremely important to predict the pathogen contamination. Therefore, we further developed an indirect competitive mode E.coli O157 H:7 detection system with using a fluorescence-labeled aptamer that specifically binds to E.coli O157 H:7 whole cells. In consideration of the complex matrix in environmental water samples and the high potential non-specific binding or adsorption on sensor surfaces, we proposed and employed an indirect detection mode to overcome these challenges. Additionally, the non-immobilized aptamers provide much more relax binding between E-coli cells and aptamers, and also shorten the binding time. The biosensor's sensing time, sensitivity, specificity, resistance to background interference and reusability were evaluated. The developed portable E.coli O157 H:7 sensing system exhibits a sensitive response concentration range ( 10 to 106 cfu/ml) and detection limit (10 cfu/ml) comparable or superior to the previously reported biosensors or other analytical methods. The portable biosensor is therefore potentially applicable for real-time and even on-site analysis of pathogen indicator in water without any complicated pre-concentration and pre-treatment steps. At last, we developed a novel portable optical fiber sensor platform-based biosensor for the detection of adenovirus via using an anti-hexon antibody for specifically binding adenovirus hexon proteins. It is known that adenovirus infection, which is a waterborne viral infection, is one of the most important causes of human morbidity in the world. Current traditional adenovirus detection methods take days to weeks (i.e. 2 to 3 weeks for culturing and plaque counting methods). The developed sensing system is completed in less than an hour with detection limit of 3 to 7 pfu/ml at different iv conditions, comparable to the most sensitive methods reported in the literature such as PCR-based method or fluorescent cell sorting assay (FACs assay). To leverage the advances in nanodevice fabrications, we developed nanobiosensors for rapid, label-free, reusable, sensitive and selective environmental pollutant (small molecules, viruses and pathogens) detection with using the single walled carbon nanotubes (SWCTNs) based nanosensor systems. Using a newly established high-rate nanoscale offset printing process with directed assembly and transfer of nanomaterials, carbon nanotubes were assembled at the desired locations with controllable high density, alignment and good uniformity that enables more stable and reusable biosensor system. In these SWCNT-based nanobiosensors systems, biorecognition probe molecules (probe-DNA for aptamers and G-quadruplex, and antibody for adenovirus) were first immobilized on the SWCNTs surface by non-covalent functionalization which leads to advantages for stable and sensitive detections. The two gold electrode terminals and SWNTs-bridge between them allow continuous and direct conductance/resistance response monitoring for the various environmental pollutants detection. Carbon nanotubes have been demonstrated as an ideal candidate for sensing due to their extremely high surface-to-volume ratio, exquisite conductance and surface chemistry. Compared to other nanomaterials, SWCNT has been the most investigated for sensor development because of its relatively low cost, high mechanical strength, easy-to-functionalize surface chemistry and unique electronic properties. The conductance of SWNT is highly sensitive to the single molecular adsorption and reaction on its surface as a result of one-dimensional current flow extremely close to its surface. Compared to previously reported SWCNT-based biosensors that use FETs (field effect transistors), our system is simpler and cost effective in fabrication steps and has quite similar sensitivity and fast response capability with FETs. Thus, the developed SWCNTs based nanobiosensor system is quite suitable for sensitive, rapid and cost effective environmental pollutants detection. Using this SWCNTs nanobiosensor system, a novel aptamer-based SWCNTs biosensor was developed for Oxytetracycline (OTC) detection which is a widely used antibiotic in agricultural areas and can accumulate in most foods (i.e. milk, eggs, vegetables etc.). Ultimately, this accumulation is likely to have serious implications for human health. Thus, simple, fast and easy to use methods are needed for OTC detection. The newly developed OTC detection will be an example of small molecule detection with using SWCNTs based nanobiosensor in real waste water samples. A label-free DNA sensor for the detection of lead ions (Pb2+) based on lead (II)-stabilized G-quadruplex formation is also demonstrated in this nanobiosensor platform. The detection sensitivities (2.5 nM for OTC and less than 1 nM for Pb2+) are greatly improved compared with most of the previous analytical detection systems and biosensors for Oxytetracycline and Pb2+ detection. Additionally, the developed nanobiosensor systems for both contaminants have improved reproducibility, reusability and stability (up to 30 days for more than 100 reusage). We also explored and developed SWCNT nanobiosensors for direct and rapid adenovirus detection, which has not been reported in the literature. This label free SWCNT-based nanobiosensor can detect adenovirus as low as 5 pfu/ml, which is lower than most previously reported sensor systems. Moreover, the developed sensing system greatly reduced the detection time length (less than 30 minutes) and the cost compared to the conventional plate counting methods. v Lastly, highly sensitive aptamer-based SWCNTs biosensor containing probe-DNA (complementary of E.coli O157 H:7 aptamer) immobilized on functionalized SWCNTs was developed for E.coli O157 H:7 detection in an indirect competitive detection mode. This conductance based nanobiosensor can detect E.coli O157 H:7 in less than 45 minutes ( including pre-incubation and regeneration steps) with the detection limit of 4 cfu/ml, which is comparable or better than previous biosensors, PCR, and traditional colony counting methods. Similar to the optical fiber biosensor, the developed indirect detection mode sensing provides much more relax binding between E-coli cells and aptamers, and also shortens the binding time. In most previous reported biosensors system, the regeneration or reusability, and real environmental sample matrix interferences were hardly evaluated and reported. For all our sensor systems, we evaluated the regeneration, stability and reproducibility of the sensing systems, as well as the potential interference from real waste water or natural water samples. We have demonstrated that both optical fiber and SWCNT based nanobiosensor systems can be reusable with proper regeneration steps, they exhibit good reproducibility and stability (up to 30 days) and robust against potential interference from soluble substances in the environmental samples. However, particulates in the environmental sample may pose interference, and therefore a simple filtration or microfluidic base separation of particles from environmental samples may be needed for the field applications.
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