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Title: Bacterial detection using an anharmonic acoustic aptasensor
Author: Khobragade, Shilpa
ISNI:       0000 0004 7970 7438
Awarding Body: Loughborough University
Current Institution: Loughborough University
Date of Award: 2018
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Infectious diseases are currently one of the greatest global challenges in medicine. Rapid and precise diagnosis and identification of pathogen is important for timely initiation of appropriate antimicrobial therapy. However, many patients with infectious diseases receive empirical treatment rather than appropriate pathogen-directed therapy. As a result antimicrobials have been overused and/or misused, which has ultimately led to antimicrobial resistance (AMR). AMR is broadly considered as the most significant public health threat facing the world today. Policy makers from all over the world have recognised the urgent need for rapid point-of-care (POC) diagnostics that would not only identify pathogens but also provide antimicrobial susceptibility profiles in meaningful timeframe to initiate appropriate antimicrobial therapy and thereby prevent AMR. Traditional culture-dependent diagnostic methods are still considered as gold standard methods. But they are very slow and generally require 18 to 48 hours with further 8 to 48 hours to perform antibiotic susceptibility test. Among culture-independent methods, PCR and ELISA are label-based, costly, laborious and require specialised equipment and trained personnel to operate them. Lateral flow assays (LFAs) that are low-cost, simple, rapid and paper-based portable detection platforms are very popular as they can be applied at the POC. However, it is difficult to integrate them with electronics and they also suffer from higher false-positive rates than PCR and ELISA. Optical, electrochemical and acoustic biosensor-based methods allow label-free, whole-cell bacterial detection. Among them, a relatively new technique called Anharmonic acoustic Detection Technique (ADT) is integrable and can be applied at the POC for rapid, label-free, low-cost, detection. ADT also allows detection with high specificity, which is lacking in most of the other methods; therefore, it was selected to explore its use for whole-cell bacterial detection. For the first time, ADT was integrated with specific multivalent DNA aptamers that bind whole bacterial cell for direct detection and quantification of viable E. coli (KCTC 2571) bacteria. This aptamer-based assay coupled with ADT constituted the anharmonic acoustic aptasensor. DNA aptamers were immobilised through biotin and streptavidin conjugation, onto the gold surface of quartz crystal resonator (QCR) to capture the target bacteria and the detection was accomplished by measuring the shift in the maximum value of magnitude of the transduced 3fsignal (3 times the drive frequency) i.e. ΔI3fmax upon binding, when driven at fundamental resonance frequency. This aptasensor provides a practical method for rapid, sensitive and quantitative on-site detection of bacteria confidently up to 2.5 x 10⁴ (corresponding to 10⁶ E.coli/mL) in 5 min, as compared to standard frequency shift (Δf₀)and dissipation shift (ΔD) measurements. Anharmonic acoustic aptasensor also demonstrated specificity by distinguishing between specific (E.coli) and non-specific (S.typhi) interactions. Linear quantitative correlation could be reliably achieved with 3fsignal (R²= 0.984) for four concentrations of E.coli bacteria (10⁵-10⁸ cells/mL), than with Δf₀ and ΔD. Comparative performance of other bio-receptors of different size (anti-E.coli antibody) and different length (shorter linker with thiolated aptamer) was assessed against longer linker with biotinylated aptamer which gave the highest sensitivity and specificity than these two different receptors. To further enhance the sensitivity and specificity of this system, the same E.coli-binding aptamers were modified by introducing smart chemical moieties into their structure such that the newly configured aptamer sequences not only quantitatively detected bacteria but also gave characteristic signal, highly specific for that interaction. Thus, this anharmonic acoustic aptasensor can be applied for rapid, sensitive, specific and quantitative detection of whole-cell bacteria and thereby, can reduce diagnostic cycle and improve the appropriate prescribing of antimicrobial therapy and thus can be used to combat AMR.
Supervisor: Not available Sponsor: Loughborough University
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Mechanical Engineering not elsewhere classified ; Antimicrobial resistance ; Anharmonic detection ; Acoustic sensor ; Aptasensor ; Rapid point-of-care diagnostics