Development of novel aflatoxin B1 biosensors by carbon nanotube integrated microfluidic systems

Abstract

Aflatoxin B1 (AFB1), which contaminates food and feed via molds, is carcinogenic to humans and animals. In addition to adverse effect on human and animal health, it results in product losses due to the difficulties of decontamination. When negative health consequences and product losses are studied economically, it is clear that they result in significant losses and issues for developing countries. To preserve human and animal health, as well as prevent product and economic losses, AFB1 contamination must be regulated at all phases of the food production system, from field to fork. Effective risk assessment and risk management techniques are required to prevent food contamination. Risk assessment requires scientific methodologies. The first stage is to use scientific procedures to detect, measure, or analyze. Traditional AFB1 detection/analysis methods such as microbiological cultivation, chromatographic analysis and ELISA test involve complicated laboratory procedures and difficult, long processes requiring expert personnel. It is expensive because it requires a lot of sample and chemical solvents, and causes environmental pollution. It forces our country to be dependent on foreign sources due to the import of analytical devices. It is conceivable to reduce such long and complex procedures into a chip and realize the lab-on-chip (LOC) concept with the ultimate technology of microfluidic systems. Today, with the advancement of microfluidic technology, it is possible to actualize such long and complex laboratory operations by miniaturizing them to a chip, thereby bringing the notion of laboratory-on-chip (LOC) to reality. In our country, it is vital to profit from the advantages of such advanced technology in items with competitive power in exports, such as olive oil. Where several governmental entities have taken measures toward quality improvement and branding by resolving existing problems, and comprehensive R&D studies must be conducted to address such issues. The submitted thesis describes the development of an integrated microfluidic system that extracts, detects, and quantifies AFB1 in olive oil by combining microfluidic technology and nanotechnology. The integrated system will include three modules: a paper-based microfluidic AFB1 biosensor (µPAD), a PDMS-based microfluidic mixer connected to µPAD, and a syringe pump system for introducing AFB1-treated olive oil and extraction solvents at specific times and volumes. The aim of the thesis is defined as; i. Creating AFB1 detection areas using different techniques with the help of hydrophobic materials on cellulose-based filter paper, ii. Functionalization of MWCNTs by immobilizing antibodies that will specifically identify the target AFB1, iii. Integration of detection areas of functionalized MWCNTs, iv. Production and optimization of PDMS-based microfluidic mixers, v. Integration of microfluidic biosensor and mixer with syringe pumps, vi: Extraction of AFB1 from olive oil with microfluidic mixers, vii. Validation of the microfluidic biosensor and quantitative measurement of extracted AFB1 samples. The first stage of the thesis is to create detection areas and channels on filter papers with the help of various hydrophobic materials. For this purpose, different fabrication techniques were applied for each hydrophobic material. When variables such as ease of manufacture, cost, analysis in which the biosensor will be utilized, and required chemical solvent resistances were considered, it was decided to continue experimental research with the wax dipping approach. The second stage is to transform the detection areas created on paper into biosensor surfaces where we can perform AFB1 detection and quantitative analysis. At this stage, nanotechnology methods were utilized. Multi-walled carbon nanotubes (MWCNT) are used in biosensors because they improve detection sensitivity by increasing surface area and provide an ideal surface for the immobilization of biological components such as antibodies. For biological detection, MWCNT surfaces must be biologically functionalized and distributed uniformly in a dispersant to ensure homogenous integration in detecting areas. Chemical alterations must be made to the surface structures of MWCNTs to achieve both goals. Due to strong van der Waals interactions, MWCNTs disperse as bundles in water, which limits their biosensor applications. MWCNTs are distributed homogeneously in water due to the carboxyl (-COOH) and hydroxyl (-OH) groups formed as a result of the treatment of MWCNTs with chemicals such as acids or peroxides. Additionally, these groups enable antibodies to bind to the MWCNT surface. With this aim, MWCNT solutions were prepared with HNO3, H2SO4 + HNO3, H2O2, H2O2 + H2SO4 to observe the formation of -COOH and -OH groups, and the FTIR method was used to determine if the appropriate functional groups developed on MWCNTs as a result of certain time and temperature applications. According to FTIR measurement results, acid oxidations were continued with HNO3, which provides the formation of -COOH groups. XRD measurements were performed for structural analysis. In the XRD spectrum of MWCNT functionalized with HNO3, the MWCNT crystal plane with added carboxyl groups was seen at 260 and the MWCNT characteristic crystal plane was seen at 430. The presence of the -COOH group was examined by XPS measurements. According to the results obtained from the elemental compositions, the Oxygen (O) ratio increased as a result of the treatment of MWCNTs with acids. Oxygen originates from -COOH groups formed as a result of reactions with acids on the surface. Nitrogen (N) was seen as a result of antibody immobilization, indicating that protein was added to the system. The increase in Oxygen (O) ratio along with Nitrogen (N) shows that protein structures are immobilized on the surface. In other words, the antibody to which AFB1 will specifically bind is immobilized on the MWCNT surface. As understood from the morphological characterizations, there was no significant change occurred in MWCNT morphologies after HNO3 treatment, and their structures were not damaged. With antibody immobilization, a beaded appearance like a cover was formed on the MWCNT surface. This shows that the protein layer bound to MWCNTs was formed and the immobilization was successful. A paper-based biosensor (µPAD) was successfully created by dispersing antibody-functionalized MWCNTs in water and integrating them onto the paper detection areas obtained in the first stage via the drop casting technique. In order to obtain sensitive results in food contamination analyses, the contaminant must be extracted from the food matrix. The other part of the thesis work is AFB1 extraction from olive oil. PDMS-based microfluidic mixers were fabricated for pre-teratments. Separation and mixing abilities were observed with various designs such as straight, sunflower, 1800 and 2400 degree bend angles, 200, 500, and 1000 µm channel widths. After COMSOL simulations and experimental optimizations with different microchannel designs, it was decided to fabricate microfluidic mixers with sunflower geometry. PDMS based mixers were created via UV litography and soft litography techniques. PDMS was preferred as the second surface instead of glass due to its flexibility and ease of micromixing. The last phase of the thesis consists of detection and quantitative analysis studies with µPADs. In AFB1 detection studies, it was observed that AFB1 could be detected at a concentration of 1 ng/mL under the FL microscope, thanks to the FL dye-labeled secondary antibody. Quantitative studies performed with a FL microscope were carried out with AFB1 standard solutions. Standard solutions prepared in the concentration range of 0.01-100 ng/mL were dropped onto the µPAD surface and examined under a microscope. Because clear results could not be obtained from MWCNT alone, silver nanoparticles (AgNP) were introduced to the system during the functionalization stage. Thanks to AgNPs, quantitative measurements were made by image analysis and a calibration graph was created. The lowest detectable concentration was determined as 0.1 ng/mL. Resistance measurements (four point probe) were made on MWCNT surfaces with the same standard AFB1 solutions and a calibration graph was created. The lowest detectable concentration was determined as 0.01 ng/mL. More precise measurements were made with resistance measurements. Both types of studies have shown that µPADs have two different linear working ranges: 0.1-1 ng/mL and 1-10 ng/mL. Microfluidic mixers integrated with syringe pumps were used for pre-teratment studies. After AFB1 spiked olive oil samples passed through the microfluidic mixer, quantitative resistance measurements were conducted. The biosensor recovery rates ranged from 91 to 97%. FL spectroscopic measurements in AFB1 standard solutions served to validate the results. Linear operating range and recovery rates are likewise within acceptable levels, according with the literature. The integrated system has high sensitivity AFB1 detection capacity. It has the potential to be used in every field where AFB1 detection is legally required. When the lowest quantities found are studied, it is clear that detection may be accomplished with a sensitivity below the legal limits. It will be cheaper compared to other technologies due to the materials used, it will reduce the economic burden required by the analysis since it is sufficient to use less chemicals and samples, and it will provide an environmentally friendly technology due to less waste chemicals. AFB1 can be used not only in the laboratory environment but also without the need for expert personnel because it is portable and simple to use. It is a modular analysis system that will enable on-site detection. In addition, it is a domestic product and domestic patent technology that will reduce our country's import dependence on analytical devices and technologies. Due to the combination of microfluidic technology and nanotechnology, the system has many differences and advantages compared to academic studies, patents and commercial products that serve similar purposes. It has the potential to turn into a domestic and high value-added product that has the power to create its own market in the national and international arena if it is patented and commercialized with its originality.