PREPARATION, CHARACTERIZATION AND STUDIES OF PHYSICOCHEMICAL AND BIOLOGICAL PROPERTIES OF DRUGS COATING LACTOSE IN FLUIDIZED BEDS

Objective: Study physicochemical properties and activity of biotechnological drugs coating lactose particles in fluidized beds for the development of a prospective approach of their identification. Methods: Lactose monohydrate as pharmaceutical excipient; affinity-purified polyclonal rabbit antibodies to recombinant human interferon-gamma as a drug substance; Pilotlab fluid bed apparatus was used for lactose powder saturation with solutions of pharmaceutical substances to the point of granulation (pelletizing); inverse light scattering method (2D-LS) for analysis of micron vibrations frequency spectra of samples surfaces for light intensity distribution in time by values of d1, d2, d3 primary descriptors; low angel and dynamic laser light scattering (LALLS, DLS) methods for distribution of lactose-water (LW) supramolecular complexes into volume fractions (micron "size spectra"), using the Master Sizer 2000 instrument and Zeta Sizer Nano ZS instrument in the nanoscale; Spirotox method for research of biological activity to determine the activation energy (Ea) values of cell death in solutions of tested samples. Results: Changes in 2D-LS scattering time on sample surfaces, described by topological descriptors, made it possible to clearly differentiate the intact lactose from fluidized samples by specific corridors in coordinates di=F(t). The calculated activation energy (Ea) values of cell biosensor death process in solutions of drugs coating lactose allow to characterize the biological activity of it in the initial (by Ea increase) and activated state (by Ea decrease) after the creation of intra-laboratory transmucosal conditions. A unique dimensional spectrum of LW complexes in the nanoscale range was obtained by DLS. The differences between samples in the distribution of LW complexes in the size range from 1 µm to 125 µm was showed by LALLS. Conclusion: The developed approach, including Сhemometrics, laser and biotesting methods can be used for qualitative the analysis tasks as well as for analytical control of the fluidization process in cases where identifiable pharmaceutical substances are not distinguishable by traditional analytical methods

Immunosensors

Immunosensors are solid-state devices in which the immunochemical reaction is coupled to a transducer. They form one of the most important classes of affinity biosensors based on the specific recognition of antigens by antibodies to form a stable complex, in a similar way to immunoassay. Depending on the type of transducer there are four types of immunosensor: electrochemical, optical, microgravimetric and thermometric. The most commonly used bioelements for the development of electrochemical immunosensors are antibodies (Ab), followed by aptamers (Apt) and, in the last five years, microRNA (miRNA). In order to perform an early diagnosis, a method that is able to measure peptides and proteins directly in a sample, without any sample pre-treatment or any separation, is preferred. This direct detection can be performed with methods making use of the specific interaction of proteins with Ab, Apt and miRNA. The recent developments made in the immunosensor field, regarding the incorporation of nanomaterials for increased sensitivity, multiplexing or microfluidic-based devices, may have potential for promising use in industry and clinical analysis. Some examples of assays for several commercially available biomarkers will be presented. The main application fields, beside biomedical analysis, are drug abuse control, food analysis and environmental analysis

Graphenated organic nanoparticles immunosensors for the detection of TB biomarkers

Magister Scientiae - MSc (Chemistry)Pulmonary Tuberculosis (TB) a disease second to HIV/AIDS is a global health problem that arises in two states; as an active state and as a latent state. Diagnosis of active TB is tedious and requires expensive procedures since there is no recognizable method for the sole detection of active TB. The current diagnosis consists of chest X-rays and multiple sputum cultures used for acid-fast bacilli detection. The TB diagnosis of children is particularly difficult which further complicates the diagnosis. Thus, rapid identification of this pathogen is important for the treatment and control of this infection to allow effective and timely therapy. In an effort to solve this issue, this study reports the development of immunosensors constructed with electroactive layers of amino groups functionalized graphene oxide (GO) doped respectively with green synthesized zinc oxide (ZnO NPs) nanoparticles and silver (Ag NPs) nanoparticles on glassy carbon electrodes. The surface morphology of GO, ZnO NPs, Ag NPs and their composites was revealed by employing High-Resolution Transmission Electron Microscopy (HR-TEM) and High-Resolution Scanning Electron Microscopy (HR-SEM) while the composition and structure of these materials were studied using Fourier Transform Infra-Red Spectroscopy (FTIR). The resultant graphene oxide-metallic composites were covalently attached with CFP-10 and/or ESAT-6 antibodies to achieve the electrochemical detection. The immunosensor was then used for the impedimetric and amperometric detection of anti-CFP-10 and/or anti-ESAT-6 antigens in standard solutions

Aptamer-based SPR Biosensor for Detection of Avian Influenza Virus

Rapid and specific detection of avian influenza (AI) virus is urgently needed with the concerns over the outbreaks of highly pathogenic H5N1 avian influenza in animal and human infection. Aptamers are artificial oligonucleic acid that can specifically bind to target molecules. They show comparable affinity for target virus and better thermal stability than monoclonal antibodies. Those advantages make aptamers promising candidates in diagnostic and detection applications. The goal of this research was to use DNA&ndashaptamer as the specific recognition element in a portable surface plasmon resonance (SPR) biosensor for detection of AI H5N1 virus in poultry. A SPR biosensor was fabricated using the selected aptamers based on streptavidin&ndashbiotin method. Streptavidin was directly adsorbed onto the surface of a gold waveguide in the SPR biosensor. Then, biotinylated aptamers were immobilized on the sensor surface via streptavidin&ndashbiotin binding. The immobilized aptamers captured AI H5N1 virus in a sample solution, causing an increase in refraction index (RI). Performances of the aptamer&ndashbased SPR biosensor were studied in streptavidin modification, aptamer immobilization and virus detection. The optimal concentrations of streptavidin and aptamers were determined to improve the sensitivity of the biosensor. The response of the aptamer&ndashvirus interaction was shown to be virus titer&ndashdependent, and a linear range for the titers of AI H5N1 was found between 0.128 and 1.28 HA unit. The aptamer&ndashbased SPR biosensor could detect the H5N1 virus at a titer greater than 0.128 HA unit within 1.5 h. No significant interference was observed from non&ndashtarget subtypes such as AI H7N2, H9N2, H2N2, H1N1 and H5N2. The aptamer&ndashbased SPR biosensor was further evaluated for detection of AI virus in poultry swab samples. All of the AI viruses used in this study were killed ones to ensure biological safety

Development and Modeling of a Biosensor Platform using AlGaN/GaN HEMT Devices

The history of biosensors began in 1962 with the invention of enzyme electrodes by Leland C. Clark. Since then, biosensors have come a long way with simultaneous contributions in various fields such as biology, chemistry, material science, electronics, physics and VLSI. With the advancement in science and technology, smaller, more sensitive and dependable biosensors have become a reality. Still the need for cost-effective, sophisticated, reliable, robust biosensors that can be used to detect multiple types of biomolecules remains a technological challenge to be resolved. The proposed AlGaN/GaN High Electron Mobility Transistors (HEMTs) have excellent prospect to become the biosensor platform of the future, as is investigated in this work in contrast to other types of biosensors. These devices excel over their silicon counterparts because of their inherent properties such as chemically stable bulk and surface properties and the availability of high-density two-dimensional electron gas (2DEG) at the hetero-interface which allows a highly sensitive detection of the surface-charge related phenomena. Using a floating gate configuration, only the drain current changes pertinent to biomolecule immobilization are observed. The test results are correlated with an analytical model which provides insight into the device physics. The high mobility and sensitivity inherent in its material system as well as device structure, robustness due to wide bandgap and system-level advantages make it the ultimate choice as a biosensor platform

Silica Coated Core-Shell Quantum Dot-based Electro-Immunosensor for Interferon Gamma TB Disease Biomarker

>Magister Scientiae - MScTuberculosis (TB) is a disease that results from infection by Mycobacterium tuberculosis, which is regarded the most common infecting organism. TB has killed countless numbers of people particularly in underdeveloped countries. TB bacteria can remain inactive or in dormant state for years without causing symptoms or spreading to other subjects, but as soon as the immune system of the host becomes weakened, the bacteria become active and infect mainly the lungs along with other parts of body. TB cases are further aggravated by other illnesses that affect the immune system, such as human immune virus (HIV), which is very prevalent in resource-poor countries. Interferon-gamma (IFN-γ) is a TB biomarker that has found to have all the qualities that are needed to help and cure Tuberculosis disease. Early diagnosis and treatment are essential measures for effectively controlling the disease. Traditional microbial culture-based tests are the most common methodologies currently used. Usually, these methods involve cell culture, cell counts, and cell enrichment, but this process is time-consuming and laborious, especially for the slow-growing bacteria like M. tuberculosis. Sputum smear is one of the methods currently used to detect acid fast bacilli (AFB) in clinical specimens or fluorescent staining. It is a cost-effective tool for diagnosing patients with TB and to monitor the progress of treatment especially in developing countries. The traditional method of inoculating solid medium such as Lowerstein-Jensen (L-J) or 7H10/7H11 media is also used currently it is slow and takes 6-8 weeks of incubation to diagnose the infection and further more time to determine the susceptibility patterns. The microscopic observation drug susceptibility (MODS) assay they are also used currently they rely on light microscopy to visualize the characteristic cording morphology of M. tuberculosis in liquid culture. MODS has shorter time to culture positivity (average 8 days) compared with LJ medium (average ~26 days), they are very expensive. The Gen-Probe assay specific for M. tuberculosis complex is a rapid detection that is also used, nucleic acid amplification (NAA) test results can be obtained as fast as in two hours (provided if a positive culture is present); it also has a high sensitivity of 99% and specificity of 99.2%. It holds the disadvantage of needing of positive culture that can take several days. Enzyme-linked immunosorbent assay (ELISA), is a test that uses antibodies and colour change to identify a substance. ELISA is an assay that uses a solid-phase enzyme immunoassay (EIA) to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample. It can be used to detection of Mycobacterium antibodies in tuberculosis. The Amplified Mycobacterium Tuberculosis Direct Test (AMTDT) is used for the detection of M. tuberculosis it enables the amplification and detection of M. tuberculosis rRNA directly from respiratory specimens. The diagnostic methods employing genetechnology based on the amplification of DNA or RNA are expected to improve the speed, sensitivity, and specificity of Mycobacterium tuberculosis detection. TB rapid cultivation detection technique, such as MB/BacT system, BactecMGIT 960 system and flow cytometry. The BACTEC MGIT960 system (Becton Dickinson, Sparks, MD) performs incubation and reading of the tubes continuously inside the machine using a predefined algorithm to interpret the fluorescent signal and giving the results as positive or negative. When performing DST, the BACTEC MGIT960 interprets the results as susceptible or resistant to the antibiotic under study. Results are available within 8 days. A recent meta-analysis of the published studies found high accuracy and high predictive values associated with the use of BACTEC MGIT960. These methods are more sensitive and rapid than the traditional microbial culture-based methods. However, they cannot provide the detection results in real-time and most of these methods are centralized in large stationary laboratories because complex instrumentation and highly qualified technical staff are required. Recently, Food and Drug Administration (FDA) approved two new assays that were introduced. These two assays detect in vitro a specific immune response to M. tuberculosis. These tests are the QuantiFERON-TB Gold In-Tube (Cellestis/Qiagen, Carnegie, Australia) and the T-SPOT.TB assay (Oxford Immunotec, Abingdon, United Kingdom). Both assays use whole blood from the patient and measure the production of interferon gamma after the whole blood is exposed to specific antigens from M. tuberculosis. These tests are based on the knowledge that IFN-γ is a product of an active cell-mediated immune response induced by M. tuberculosis. However, TB detection remains a major obstacle due to several drawbacks of these methods. To date, the number of diagnosis approaches for TB has increased as the disease continues to be a major public health problem worldwide and most conventional detection technologies present difficulties in recognizing the presence of M. tuberculosis, since they are time consuming, do not provide clinically reliable results and significantly lack of sensitivity. This thesis focusedon developing two binary and one ternary-electrochemically quantum dots, all synthesised at room temperature in aqueous media for detecting (IFN-γ). Copper telluride (CuTe) and Zinc telluride (ZnTe) was prepared to check how does the two quantum dot behave individual and also to check on how they behave when they are combined and formed ternary quantum dots (CuZnTe). The electrochemical studies of the binary CuTe quantum dots, ZnTe quantum dots and the ternary CuZnTe core-shell quantum dots reveal that ternary quantum dots were stable and showed a significant enhancement in the conductivity of CuZnTe core-shell solution compared to that of CuTe and ZnTe, all studied in solution. The three different quantum dots were capped with three different capping reagents which are tetraethyl orthosilicate (TEOS), thioglycolic acid (TGA), (3-mercaptopropyl) trimethoxysilane (MPS). In the study, a label-free electrochemical immunosensor for the detection of interferon gamma (IFN-γ) was prepared for the first time using ternary quantum dots. The biosensor consists of water-soluble silica coated Copper Zinc telluride (CuZnTe core-shell) quantum dots conjugated to a gold electrode. The antibody-antigen were then conjugated on the CuZnTe core-shell QD modified gold electrode. Results from synthesis of two different binary quantum dots are also presented in the study and compared to the results of the CuZnTe core-shell QDs. The CuTe quantum dots had a small average size which was confirmed through HRTEM, SAXS and XRD analysi

Biomarkers Used for the Diagnosis of Diseases

The detection and quantification of with high precision nucleic acid biomarkers and protein biomarkers in resource-limited settings is key to the early diagnosis of diseases and for monitoring the effects of treatments. As there is an enormous demand for high-quality biomarker detection platforms that are robust and highly applicable in resource-limited settings, this book is devoted to exploring methods for detection and quantification of biomarkers, focusing on the recent advances in this field

ALTERNATING CURRENT ELECTROKINETICS BASED CAPACITIVE AFFINITY BIOSENSOR: A POINT-OF-CARE DIAGNOSTIC PLATFORM

Capacitive bioaffinity detection using microelectrodes is considered as a promising label-free method for point-of-care diagnosis, though with challenges in sensitivity, specificity and the time “from sample to result.” This work presents an alternating current (AC)-electrokinetic based capacitive affinity sensing method that is capable of realizing rapid in-situ detection of specific biomolecular interactions such as probe-analyte binding. The capacitive biosensor presented here employs elevated AC potentials at a fixed frequency for impedimetric interrogation of the microelectrodes. Such an AC signal is capable of inducing dielectrophoresis (DEP) and AC electrothermal (ACET) effects, so as to realize in-situ enrichment of macro and even small molecules at microelectrodes and hence accelerated detection. Experimental study of the DEP/ACET-enhanced capacitive sensing method was conducted, and the results corroborate our hypothesis. This capacitive sensing method has been shown to work with various types and sizes of biomolecules (such as antibodies, virus and small molecules) to differentiate disease-positive samples from negative samples within or less than two minutes, while conventional assay would require multiple processing steps and take hours to complete. The results showed high accuracy and sensitivity. Overall, this capacitive affinity biosensor may form a basis for the development of a feasible point-of-care diagnostic platform for the detection of infectious diseases in the future

Development and evaluation of QCM sensors for the detection of influenza virus from clinical samples

The Quartz crystal microbalance (QCM) is a very sensitive mass-detecting device which is based on changes in to the vibrational frequency of quartz crystals after adsorption of substances to a modified crystal surface. In this study a QCM-based biosensor was developed for the rapid diagnosis of influenza viruses and its suitability and limitations were compared with currently available diagnostic methods on 67 clinical samples (nasal washes) received during the 2005 Australian winter. The type-specific and conserved viral M1 proteins of both A/PR/8/34 and B/Lee/40 viruses were used to prepare polyclonal antisera for the development of an ELISA. The limits of detection of ELISAs for the detection of purified A/PR/8/34 and B/Lee/40 ƒnviruses were 20ƒÝg/mL ƒnand 14 ƒÝg/mL using polyclonal antibodies, and 30 ƒÝg/mL and 20 ƒÝg/mL for monoclonal antibodies, respectively. The limit for detection of each virus was 104 pfu/mL, irrespective of whether antisera or monoclonal antibodies were used for capture. Non-purified cell culture-grown preparations of either virus could be detected at 103 pfu/mL The QCM utilised the same reagents used in ELISAs. However, a number of parameters were then further optimised to improve the sensitivity of the tests. These included blocking of non-specific binding, examination of the effects of flow-cell compression, the role of pH, flow rate, antibody concentration and the addition of protein A to the crystal surfaces of the biosensor. The lowest virus concentration that could be detected with the QCM was 104 pfu/mL for egg-grown preparations of both A/PR/8/34 and B/Lee/40, which could be detected within 30 min. However, conjugation of 13 nm gold nanoparticles to a second detector antibody resulted in a 10-fold increase in sensitivity and a detection limit of 103 pfu/mL that could be determined within 1 h. The direct detection of the influenza viruses in nasal samples was not possible by QCM because of the significant frequency fluctuation that was probably caused by the viscosity of the samples. Therefore, an additional culture step of 12 h was required, which increased the processing time to 2 days. The QCM/nanoparticle method was shown to be as sensitive as the standard cell culture method, and the QCM method as sensitive as the shell vial method. The QCM and QCM/nanoparticle methods were shown to be 81 and 87% as sensitive and both were 100% as specific as the real-time polymerase chain reaction. However, for use in rapid diagnosis, improvements are required to remove frequency fluctuation resulting from the direct use of nasal samples

Biosensor-based studies on coumarins

Polyclonal antibodies to 7-hydroxycoumarin - the main metabolite of coumarin, a plant constituent with many clinical applications - were produced, purified and characterised. This antibody preparation was used to develop a competitive immunoassay for 7- hydroxycoumarin which was carried out on the BIAcore, an optical biosensor based on the phenomenon of surface plasmon resonance (SPR). The immunoassay was optimised and internally validated. Monoclonal antibodies were generated by somatic cell fusion using the spleens of mice immunised with a 7-hydroxycoumarin-protein conjugate. The screening of cell culture supernatants by BIAcore and ELISA was compared, and all of 13 clones isolated by limiting dilution were found to be reactive to the drug-protein conjugate, but not to free drug. The panning of phage displayed antibodies from a naïve library using the BIAcore was investigated, and, although the amount of bound phage was insufficient to generate an SPR signal, subsequent analysis of eluate demonstrated that enrichment had taken place. BIAcore was also used to affinity rank a panel of 3 genetically-produced single chain Fv antibodies against coumarin-BSA and to examine kinetic and affinity data for the interaction of one of these antibodies with immobilised drug-protein conjugate. The interaction of two antibody preparations against the fungal toxin aflatoxin Bi (AFBj), a member of the coumarin family, was studied using both BIAcore and ELISA methods. High affinity constants rendered the monoclonal and polyclonal antibodies unsuitable for use in regenerable immunosensor formats. A range of competitive and sandwich ELISAs for the detection of AFBi were developed using both types of antibody. In addition, methods to facilitate better design of formats for use with BIAcore were established using ELISA to mimic regeneration conditions on the sensor chip surface, and to calculate equilibrium affinity constants. Supercoiled plasmid DNA was also immobilised on the BIAcore sensor surface, and the inhibition of the enzyme topoisomerase II by the coumarin antibiotic novobiocin, as well as the direct binding of a range of coumarin-protein conjugates to nucleic acids, were investigated