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Novel impedimetric immunosensor for detection of pathogenic bacteria Streptococcus pyogenes in human saliva
Asif Ahmed1, Jo V Rushworth, John D Wright
1School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds , Leeds LS2 9JT, U.K.
Abstract:
Streptococcus pyogenes , also known as group A streptococcus (GAS), is a Gram positive human pathogen responsible for invasive and noninvasive human infections with a high incidence rate. Traditional detection methods involve cell culture and PCR, which are limited by long processing times or the need for high cost equipment. Impedance-based electrochemical immunosensors provide an alternative by which precise and rapid quantitative detection of the organism can help with rapid clinical decisions. To bring a biosensor for point-of-care applications to market, strict optimization of each level of construction and operation is required. In this paper, commercial screen-printed gold electrodes have been used to construct polytyramine (Ptyr)-based immunosensors. Biotin tagged whole antibodies against S. pyogenes were conjugated to Ptyr amine group via biotin-NeutrAvidin coupling. Sensors were optimized at each level of construction, particularly for Ptyr electrodeposition and antibody concentration, to optimize signal and specificity. Scanning electron microscopy, fluorescence microscopy, and on-sensor analysis (HRP conjugated enhanced chemiluminescence-based semiquantitative method) to detect Ptyr surface amine and bound antibody were performed as supporting techniques. Cumulative and single shot incubations had shown detection range of 100 to 10(5) cells per 10 μL and 100 to 10(4) cells per 10 μL of bacteria in PBS, respectively. Sensors were also able to specifically detect S. pyogenes in 50% (v/v) human saliva, with good selectivity and low cross-reactivity.
Insights
This study presents a novel electrochemical immunosensor for rapid and precise detection of Streptococcus pyogenes. The developed biosensor offers sensitive and specific identification of bacteria in various samples, aiding faster clinical decisions.
Area of Science:
- Biomedical Engineering
- Biosensor Technology
- Microbiology
Background:
- Streptococcus pyogenes (group A Streptococcus) is a significant human pathogen causing widespread infections.
- Traditional detection methods (cell culture, PCR) are time-consuming and require expensive equipment.
- Rapid and accurate diagnostics are crucial for effective clinical management of GAS infections.
Purpose of the Study:
- To develop and optimize an impedance-based electrochemical immunosensor for rapid, quantitative detection of Streptococcus pyogenes.
- To enable point-of-care applications by optimizing sensor construction and operation.
- To assess the sensor's performance in terms of sensitivity, specificity, and detection range.
Main Methods:
- Construction of polytyramine (Ptyr)-based immunosensors on commercial screen-printed gold electrodes.
- Conjugation of biotin-tagged anti-S. pyogenes antibodies to Ptyr via biotin-NeutrAvidin coupling.
- Optimization of Ptyr electrodeposition and antibody concentration, validated by microscopy and chemiluminescence assays.
Main Results:
- The immunosensor demonstrated a detection range of 100 to 10^5 cells/10 μL (cumulative) and 100 to 10^4 cells/10 μL (single shot) in PBS.
- Specific detection of S. pyogenes was achieved in 50% (v/v) human saliva.
- The sensor exhibited good selectivity and low cross-reactivity with other substances.
Conclusions:
- The developed polytyramine-based electrochemical immunosensor offers a promising platform for rapid and sensitive detection of Streptococcus pyogenes.
- This biosensor technology has the potential for point-of-care applications, facilitating quicker diagnosis and treatment.
- Further optimization can enhance its utility in clinical settings for managing GAS infections.
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