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Fast and sensitive detection of pathogenic microbial cells is a highly important task in medical diagnostics, environmental analysis and evaluation of food safety. Accordingly, the idea of microorganism identification by the recognition of specific DNA sequence using electrochemical technique is one of the leading researches in the development of diagnostic devices. In other words, it should only be a matter of time before a small portable electrochemical device is available at the care centre for quick diagnoses of a patient’s infectious disease. However, bottlenecks for such diagnostic systems include selectivity, sensitivity, automation, sample pre-treatment and the architecture of miniaturization. In this thesis, a novel, highly sensitive and automated flow-based DNA-sensor technique for the detection of specific bacterial DNA sequences is introduced. The technique consists of a solid gold electrode that is functionalised using a simple and cheap chemistry to capture desired single stranded DNA in a complex matrix. The technology platform is based on the change in electrical property (capacitance) upon hybridization of the desired ssDNA to a capture probe. The physical and electrochemical properties of the modified electrode surface were studied using atomic force microscopy and cyclic voltammetry in reference to the innovative capacitive DNA-sensor assay. The DNA-sensor was optimized using homo-oligonucleotides with different number of bases (15- to 50 bases in probe length). The signal amplitude was found to increase with increase in oligonucleotide length, from 15- to 25-mer. However, there was no significant difference in signal readout between 25- and 50-mer. When using sandwich hybridization the signal readout for 50-mer oligonucleotides was increased by 46 %, from 78 to 114 -nF cm-2. In addition, stability and selectivity of the DNA-sensor were investigated at elevated temperatures by applying different types of homo-oligonucleotides on the same capture probe; the sensor proved to be exceedingly stable in wide range of temperatures, from 23 to 50 oC, with selectivity (> 95 %). To demonstrate the capability of the developed capacitive DNA-sensor in a real application, a specific capture probe designed to recognise 16S rDNA of Escherichia coli and other members in the family Enterobacteriacea was used. This, to explicitly detect certain patches of E. coli 16S rDNA from a laboratory culture. The study showed that unamplified E. coli 16S rDNA could be sensitively detected at very low concentration corresponds to 10 E. coli cells per millilitre with only 15 min hybridization time. The sensor also showed a good selectivity over Lactobacillus reuteri 16S rDNA (Lactobacillaceae) from a laboratory culture. Furthermore, a new approach for pre-treatment of the bacterial DNA-sample prior to flow-based DNA-sensor analysis is demonstrated. The novel pre-treatment method utilizes a commercial single stranded DNA binding protein to efficiently stabilize the heat generated single-stranded DNA. Subsequent addition of formamide in the mixture resulted in denaturation of the protein, and hence, hybridization of the heat-generated target ssDNA to capture probe takes place. Another promising application showed for the developed DNA-sensor is the identification of Methicillin-resistant Staphylococcus aureus based on detection of the mecA gene. The study showed that the sensor adequately could detect and recover 95 % of 0.01 nM mecA gene from spiked human saliva, with a detection limit of 0.6 pM. In conclusion, the work presented in this thesis demonstrates the development of a sensitive and effective biosensor for bacterial detection based on specific DNA sequence analysis. Compared to the commercially existing techniques for bacterial detection, the developed capacitive DNA-sensor proved to be non-complex, fast and efficient. This thesis work lays the groundwork for the development of a hand-held, field-adopted DNA-sensor for on-site microbial diagnostics, useful in e.g. remote areas. A handful of diseases-causing pathogenic microorganisms claim millions of lives worldwide each year, mainly due to limited diagnostic capabilities. The lack of simple, cost-effective, sensitive and rapid on-site diagnostic tools lead to delays in the diagnoses with a subsequent lag in treatment, which in turn can result in serious complications or even loss of lives. Infectious diseases pose a serious threat to human well-being and to the economic development. For instance, the estimate economic burden of the diseases caused by antimicrobial resistant microorganisms within the European Union amounts to € 1.5 billion over all societal costs per year; in Thailand and USA the societal costs totals US$ 1.3 and 35 billion per year, respectively. Likewise, the economic cost for multi-drug resistance Tuberculosis-related deaths in Sub-Sahara Africa from year 2006 to 2015 is estimated to be US$ 519 billion. Most of the current diagnostic tools in our hospitals and clinical laboratories are capable of detecting ‘bad bugs’ (i.e. pathogenic microorganisms) when their concentration in a patient’s clinical sample is reasonably high. However, the pathogens do not reach detectable concentration until the disease has progressed and become significantly inferior. Some of the infectious diseases such as Ebola haemorrhagic fever, which has killed more than 8000 people in West Africa, need an immediate intervention to hinder the exponential spreading and to prevent the situation from becoming an even worse catastrophe than it is today. Currently, the tools for minimizing the spreading of the Ebola virus are few and merely include isolation of infected persons from the rest of the population and thereafter to simply wait out the disease. Hence, many people become infected while encountering patients during their asymptomatic stage of the disease. Although most of the current diagnostic tools are highly sophisticated, their complexity and expensive operations restrict them to off-site analysis, which additionally serve as an impediment to the efficient pathogen diagnosis in remote areas, especially in developing countries. The limited availability of on-site diagnostic capabilities has significantly increased the need for the development of rapid, cost-effective and portable devices for diagnosis of infectious diseases. In this work, a novel diagnostic device has been developed, which rapidly can detect small amount of bacteria based on the genetic material (DNA). DNA is the storage house or cellular library that contains all genetic information required in the functioning and growth of every living thing, from humans to single-cell organisms, as well as DNA-viruses. The developed device, a capacitive DNA-sensor consists of a disposable sensing chip, which is constructed using a simple and cheap manufacturing method. The capacitive DNA-sensor is automated, and it can be miniaturized and adapted for in-field use including bacterial diagnosis in remote areas under resource-limited settings. The stability and uniqueness of the DNA-material make the capacitive DNA-sensor highly effective and suitable for the targeting of specific bacteria based on their set of genes. The operational cost for the capacitive sensor is aimed to be very low as a sensing chip can be reused for at least 20 assays before replacement. Furthermore, the developed capacitive DNA-sensor is user-friendly and requires only limited amounts of training to operate. |
