Entwicklung von Einmalsensoren zur schnellen Multianalytdetektion: Acteylcholinesterase- und mikrobielle Biosensoren

Der Gegenstand dieser Arbeit war die Entwicklung von neuen Biosensoren zum simultanen Nachweis und der Klassifizierung von Stoffgemischen in der Umweltanalytik. Diese neuartigen Systeme sollten zur Detektion Acetylcholinesterase hemmender Insektizide aus der Gruppe der Organophosphate und Carbamate sowie halogenorganischer Stoffe eingesetzt werden. Diese Substanzen spielen insbesondere in der Überwachung der Wasserqualität eine entscheidende Rolle. Das Sensorprinzip basierte auf der Idee, biologische Rezeptorkomponenten, die unterschiedliche Spezifität für die Zielananlyten aufweisen, zu kombinieren, und deren Signalmuster durch künstliche neuronale Netze (KNN) auszuwerten. Alle entwickelten Systeme verwendeten durch Siebdruck hergestellte Dickschicht-Multielektroden als Sensorbasis. Zur Mustererkennung von Insektiziden wurden Biosensoren eingesetzt, welche Acetylcholinesterase-Varianten variierender Selektivität enthielten. Die Detektion halogenorganischer Verbindungen in Mischungen erfolgte mittels Sensoren, die mit Ralstonia eutropha JMP 134 Zellen bestückt waren. Die verschiedenen Selektivitäten dieser Sensoren wurden durch Kultivierung der Mikroorganismen auf unterschiedlichen Wachstumssubstraten erreicht.The objective of this work was to develop new biosensors for multianalyte detection. Target analytes were cholinesterase inhibiting insecticides and chlorinated aromatic hydrocarbons. The task was fulfilled by a combination of biological receptors bearing different selectivities for the desired analytes and data evaluation by feed-forward artificial neural networks (ANN). The basic sensor design consisted of a four-electrode thick film electrode which was fabricated by screen printing. As biological receptors for insecticide detection, variants of acetylcholinesterase (AChE) and for chlorinated aromatics Ralstonia eutropha JMP 134 cells were selected

Direct Detection and Genotyping of Klebsiella pneumoniae Carbapenemases from Urine by Use of a New DNA Microarray Test

Klebsiella pneumoniae carbapenemases (KPC) are considered a serious threat to antibiotic therapy as they confer resistance to carbapenems, which are used to treat Extended-spectrum beta-lactamase (ESBL) producing bacteria. Here, we describe the development and evaluation of a DNA microarray for detection and genotyping of KPC genes (blaKPC) within 5 hours. To test the whole assay procedure (DNA extraction + DNA microarray assay) directly from clinical specimen, we compared two commercial DNA extraction kits (QIAprep Spin Miniprep Kit (Qiagen), Urine Bacterial DNA Isolation Kit (Norgen)) for the direct DNA extraction from urine samples (dilution series spiked in human urine). A reliable SNP typing from 1×105 CFU/mL urine was demonstrated for Escherichia coli (Qiagen and Norgen) and 80 CFU/mL urine on average for K. pneumoniae (Norgen). The study presents for the first time the combination of a new KPC-microarray with commercial sample preparation for the detection and genotyping of microbial pathogens directly from clinical specimen which paves the way towards tests providing epidemiological and diagnostic data enabling better antimicrobial stewardship

Neurotoxin detection in food using disposable AChE-biosensors

The extensive use of pesticides to protect agricultural crops necessitates reliable tools for the detection of residues in food and water, thus ensuring environmental protection and consumer safety. Neuroinhibitors such as organophosphates and carbamates are widely used in agriculture because of their high insecticidal activity and their rapid mineralisation in the environment. Since they do not only inhibit insect acetylcholinesterase (AChE) but also interfere with neural transmission in other organism, including humans, they represent a potential hazard for human health and environmental food chains, and thus require continuous assessment. Recent reports on organophosphate contamination in baby food have initiated the reassessment of permitted residual concentrations of these compounds in view of the US Food Quality Protection Act. Here we present a rapid detection method for neurotoxins in food, which can increase the number of tested foodstuff and thus improve the consumer safety. We developed a highly sensitive and rapid food analysis biosensor based on disposable screen-printed thickfilm electrodes. Matrix problems were solved by using isooctane as extraction solvent. The performance of this amperometric test was checked by three different food matrixes, namely orange juice, apple and peach baby food. The detection limit was comparable to chemical standard methods and met the requirements for monitoring the maximum residue levels in baby food set by the EU

Antimicrobial resistance in patients with suspected urinary tract infections in primary care in Assam, India

OBJECTIVES: We investigated the prevalence and diversity of antimicrobial resistance in bacteria isolated from urine samples of community-onset urinary tract infection (UTI) patients in southern Assam, India. METHODS: Freshly voided midstream urine samples were collected from patients attending primary healthcare centres, with the patients’ epidemiological data also recorded. Species identification was confirmed using a VITEK 2 compact automated system. Phenotypic confirmation of ESBLs was performed using the combined disc diffusion method (CLSI 2017) and carbapenemase production was phenotypically characterized using a modified Hodge test. Common ESBLs and carbapenem-resistance mechanisms were determined in Escherichia coli isolates using PCR assays. Incompatibility typing of the conjugable plasmids was determined by PCR-based replicon typing; the phylotypes and MLSTs were also analysed. RESULTS: A total of 301 (59.7%) samples showed significant bacteriuria along with symptoms of UTI and among them 103 isolates were identified as E. coli of multiple STs (ST3268, ST3430, ST4671 and others). Among them, 26.2% (27/103) were phenotypically ESBL producers whereas 12.6% (13/103) were carbapenemase producers. This study describes the occurrence of diverse ESBL genes—bla(CTX-M-15), bla(SHV-148), bla(PER-1) and bla(TEM)—and two E. coli isolates carrying the bla(NDM-1) carbapenemase gene. ESBL genes were located within transconjugable plasmids of IncP and IncF type whereas bla(NDM-1) was carried in an IncF(repB) type plasmid. CONCLUSIONS: This study illustrates the high rate of MDR in E. coli causing UTI in primary care in rural Assam. UTIs caused by ESBL- or MBL-producing bacteria are very difficult to treat and can often lead to treatment failure. Thus, future research should focus on rapid diagnostics to enable targeted treatment options and reduce the treatment failure likely to occur with commonly prescribed antibiotics, which will help to combat antimicrobial resistance and the burden of UTIs

Amplification Free Detection of SARS-CoV-2 Using Multi-valent Binding

[Image: see text] We present the development of electrochemical impedance spectroscopy (EIS)-based biosensors for sensitive detection of SARS-CoV-2 RNA using multi-valent binding. By increasing the number of probe–target binding events per target molecule, multi-valent binding is a viable strategy for improving the biosensor performance. As EIS can provide sensitive and label-free measurements of nucleic acid targets during probe–target hybridization, we used multi-valent binding to build EIS biosensors for targeting SARS-CoV-2 RNA. For developing the biosensor, we explored two different approaches including probe combinations that individually bind in a single-valent fashion and the probes that bind in a multi-valent manner on their own. While we found excellent biosensor performance using probe combinations, we also discovered unexpected signal suppression. We explained the signal suppression theoretically using inter- and intra-probe hybridizations which confirmed our experimental findings. With our best probe combination, we achieved a LOD of 182 copies/μL (303 aM) of SARS-CoV-2 RNA and used these for successful evaluation of patient samples for COVID-19 diagnostics. We were also able to show the concept of multi-valent binding with shorter probes in the second approach. Here, a 13-nt-long probe has shown the best performance during SARS-CoV-2 RNA binding. Therefore, multi-valent binding approaches using EIS have high utility for direct detection of nucleic acid targets and for point-of-care diagnostics

Rapid Electrochemical Detection of New Delhi Metallo-beta-lactamase Genes To Enable Point-of-Care Testing of Carbapenem-Resistant Enterobacteriaceae

The alarming rate at which antibiotic resistance is occurring in human pathogens causes a pressing need for improved diagnostic technologies aimed at rapid detection and point-of-care testing to support quick decision making regarding antibiotic therapy and patient management. Here, we report the successful development of an electrochemical biosensor to detect <i>bla</i>_<i>NDM</i>, the gene encoding the emerging New Delhi metallo-beta-lactamase, using label-free electrochemical impedance spectroscopy (EIS). The presence of this gene is of critical concern because organisms harboring <i>bla</i>_<i>NDM</i> tend to be multiresistant, leaving very few treatment options. For the EIS assay, we used a <i>bla</i>_<i>NDM</i>-specific PNA probe that was designed by applying a new approach that combines <i>in silico</i> probe design and fluorescence-based DNA microarray validation with electrochemical testing on gold screen-printed electrodes. The assay was successfully demonstrated for synthetic targets (LOD = 10 nM), PCR products (LOD = 100 pM), and direct, amplification-free detection from a <i>bla</i>_<i>NDM</i>-harboring plasmid. The biosensor’s specificity, preanalytical requirements, and performance under ambient conditions were demonstrated and successfully proved its suitability for further point-of-care test development

MicroRNA-122 and cytokeratin-18 have potential as a biomarkers of drug-induced liver injury in European and African patients on treatment for mycobacterial infection

Funding: Sarah Rupprechter was funded by the UK Medical Research Council via the Doctoral Training Programme Grant in Precision Medicine at the University of Edinburgh.Aims Patients on antituberculosis (anti‐TB) therapy are at risk of drug‐induced liver injury (DILI). MicroRNA‐122 (miR‐122) and cytokeratin‐18 (K18) are DILI biomarkers. To explore their utility in this global context, circulating miR‐122 and K18 were measured in UK and Ugandan populations on anti‐TB therapy for mycobacterial infection. Methods Healthy subjects and patients receiving anti‐TB therapy were recruited at the Royal Infirmary of Edinburgh, UK (ALISTER—ClinicalTrials.gov Identifier: NCT03211208). African patients with human immunodeficiency virus–TB coinfection were recruited at the Infectious Diseases Institute, Kampala, Uganda (SAEFRIF—NCT03982277). Serial blood samples, demographic and clinical data were collected. In ALISTER samples, MiR‐122 was quantified using polymerase chain reaction. In ALISTER and SAEFRIF samples, K18 was quantified by enzyme‐linked immunosorbent assay. Results The study had 235 participants (healthy volunteers [n = 28]; ALISTER: active TB [n = 30], latent TB [n = 88], nontuberculous mycobacterial infection [n = 25]; SAEFRIF: human immunodeficiency virus‐TB coinfection [n = 64]). In the absence of DILI, there was no difference in miR‐122 and K18 across the groups. Both miR‐122 and K18 correlated with alanine transaminase (ALT) activity (miR‐122: R = .52, 95%CI = 0.42–0.61, P 50 U/L with higher sensitivity/specificity than K18. There were 2 DILI cases: baseline ALT, 18 and 28 IU/L, peak ALT 431 and 194 IU/L; baseline K18, 58 and 219 U/L, peak K18 1247 and 3490 U/L; baseline miR‐122 4 and 17 fM, peak miR‐122 60 and 336 fM, respectively. Conclusion In patients treated with anti‐TB therapy, miR‐122 and K18 correlated with ALT and increased with DILI. Further work should determine their diagnostic and prognostic utility in this global context‐of‐use.Publisher PDFPeer reviewe