Development of an Ultrasensitive Capacitive DNA-sensor: A promising tool towards microbial diagnostics
Abstract: 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.
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