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Abstract: Klebsiella pneumoniae is well-known gram-negative type opportunistic pathogen bacteria normally found in human intestine (where they do not cause disease) and responsible for different types of bacterial infections such as bloodstream infection, pneumonia, wound site infection, urinary tract infection, sepsis etc. [1,2]. Hence it is essential to develop a technique that can detect the level of bacteria. Here, we report a reliable, robust and accurate detection method using a Polypyrrole (PPy) based molecularly imprinted polymer (MIP) electrodes on ITO coated glass. This sensor was fabricated by the oxidative polymerization of pyrrole using ferric chloride oxidant in the presence of the template i.e. Klebsiella pneumoniae and subsequent removal of the template using sonication and washing with deionized water followed by electrophoretic deposition on ITO coated glass electrode. The sensing was done by the electrochemical sensing method and detection was carried out by DPV technique. The development of a PPy based electrochemical sensor that can perform the aforementioned role in monitoring its levels in any given sample. For the development of MIP mostly conducting polymers are used for entrapping the analyte due to their very good stability, speed, electrochemical properties and sensitivity [3]. In this work we only use bulk imprinting for MIP formation. In this imprinting method, bacterial cell is imprinted on the polymeric material and when polymerization process completed, the template was removed by centrifuge followed by washing with ethanol and DI to form MIP to get bacteria specific, three-dimensional interaction sites inside MIP [4-6]. The matrix PPy used for imprinting Klabseilla pneumoniae is the over oxidized PPy and carboxylic, hydroxyl and phosphate are anionic functional groups present on the outer surface of the bacterial cell wall. Thus, entrapment of bacteria into a positively charged conducting polymer simply happened because of the occurrence of linked useful microorganism groups [7]. As compared to other detection methods, MIP based electrochemical detection method offers rapid, inexpensive and selective receptors. The functional group characterization was performed using FTIR to check the incorporation and subsequent removal of template bacteria. Under the optimized conditions such as pH value, incubation time the electrochemical sensing measurements were performed using differential pulse voltammetry technique. The detection was carried out in the concentration range of 1 - 105 CFU ml-1 (Figure 1(a)) and the results showed the linear detection in the range 1 to 105 CFU ml-1 (Figure 1(b)). The developed MIP based sensor showed excellent sensitivity as 3 mA CFU-1 ml cm -2 with a limit of detection (LOD) 1.352 CFU ml-1. Further, the applicability of the nanosensor has been tested in Klebseilla pneumoniae presence in real and spiked real samples, and it was found that the nanosensor is capable enough to detect the bacteria presented in the spiked real samples. All these studies show that the developed nanosensor can be used potentially in food and environmental safety applications.
Template and target information: bacteria, Klebsiella pneumoniae
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