72502b0aa30b18fee4166cdf0f90de57 sensors_a2021v21n5p1749.pdf 5d142616b2075c3d4df9045611d1212e94d68782 sensors_a2021v21n5p1749.pdf 3b8516dfb33f7936afb1ee956f02141fe34f6cffc2554bff2084b0d12152b02b sensors_a2021v21n5p1749.pdf Title: Electrochemical Genosensing of E. coli Based on Padlock Probes and Rolling Circle Amplification Subject: Isothermal amplification techniques are emerging nowadays for the rapid and accurate detection of pathogenic bacteria in low resource settings, where many infectious diseases are endemic, and the lack of reliable power supply, trained personnel and specialized facilities pose critical barriers for timely diagnosis. This work addresses the detection of E. coli based on DNA isothermal amplification performed on magnetic particles (MPs) followed by electrochemical genosensing on disposable electrodes by square-wave voltammetry. In this approach, the bacterial DNA is preconcentrated using a target-specific magnetic probe and then amplified on the MPs by rolling circle amplification (RCA). Two different electrochemical readout methods for the RCA amplicons are tested. The first one relied on the labelling of the magnetic RCA product with a digoxigenin probe followed by the incubation with antiDIG-HRP antibody as electrochemical reporter. In the second case, the direct detection with an HRP-probe was performed. This latter strategy showed an improved analytical performance, while simultaneously avoiding the use of thermocyclers or bulky bench top equipment. Keywords: isothermal amplification; rolling circle amplification; padlock probes; electrochemical genosensing; magnetic particles Author: Alejandra Ben Aissa, Narayanan Madaboosi, Mats Nilsson and Maria Isabel Pividori Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Mar 5 08:36:50 2021 CET ModDate: Fri Mar 5 11:41:59 2021 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 13 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2545277 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- LKBNEN+VnURWPalladioL Type 1 Custom yes yes no 10 0 GOPRQR+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 LIJZZY+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 PXFODL+URWPalladioL-BoldItal Type 1 Custom yes yes no 24 0 OWRJIO+URWPalladioL-Roma Type 1 Custom yes yes no 28 0 RVSEMT+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 36 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 58 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 62 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 66 0 DYVFWR+CMSY10 Type 1 Builtin yes yes no 78 0 GOLGOE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 95 0 GOLGPE+PalatinoLinotype CID TrueType Identity-H yes yes yes 98 0 GOLGPF+PalatinoLinotype TrueType WinAnsi yes yes no 104 0 GOLHBG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 107 0 GOLGOE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 118 0 GOLGPE+PalatinoLinotype CID TrueType Identity-H yes yes yes 121 0 GOLGPF+PalatinoLinotype TrueType WinAnsi yes yes no 127 0 GOLHBG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 130 0 ZXDKIP+EURM10 Type 1 Builtin yes yes no 137 0 GOLGOE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 151 0 GOLGPE+PalatinoLinotype CID TrueType Identity-H yes yes yes 154 0 GOLGPF+PalatinoLinotype TrueType WinAnsi yes yes no 160 0 GOLHBG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 163 0 GOLGOE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 180 0 GOLGPF+PalatinoLinotype TrueType WinAnsi yes yes no 183 0 GOLHBG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 186 0 GOLGOE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 198 0 GOLGPF+PalatinoLinotype TrueType WinAnsi yes yes no 201 0 GOLHBG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 204 0 GOLGOE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 219 0 GOLGPE+PalatinoLinotype CID TrueType Identity-H yes yes yes 222 0 GOLGPF+PalatinoLinotype TrueType WinAnsi yes yes no 228 0 GOLHBG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 231 0 GOLGOE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 245 0 GOLGPE+PalatinoLinotype CID TrueType Identity-H yes yes yes 248 0 GOLGPF+PalatinoLinotype TrueType WinAnsi yes yes no 254 0 GOLHBG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 257 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-10-02 03:53:51 CEST RepresentationInformation: sensors_a2021v21n5p1749.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-03-31 11:50:38 CEST Size: 2545277 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 408 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Instrumentation Destination: subsection.2.1 Item: Title: Chemicals and Biochemicals Destination: subsection.2.2 Item: Title: Design of the Padlock, Capture and Readout Probes Destination: subsection.2.3 Item: Title: Selection of the Readout Probe Sequence and Optimization of the Rolling Circle Amplification Destination: subsection.2.4 Item: Title: Rolling Circle Amplification on Streptavidin Magnetic Particles and Electrochemical Genosensing Destination: subsection.2.5 Item: Title: Direct and Indirect Readout Approach Destination: subsection.2.6 Item: Title: Electrochemical Genosensing of E. coli. Destination: subsection.2.7 Item: Title: Results and Discussion Destination: section.3 Children: Item: Title: Selection of the Readout Probe Sequence and Optimization of the Rolling Circle Amplification Destination: subsection.3.1 Item: Title: Rolling Circle Amplification on Streptavidin Magnetic Particles and Electrochemical Genosensing. Direct and Indirect Readout Approach Destination: subsection.3.2 Item: Title: Rolling Circle Amplification on Streptavidin Magnetic Particles and Electrochemical Genosensing Destination: subsection.3.3 Item: Title: Electrochemical Genosensing of E. coli Destination: subsection.3.4 Item: Title: Conclusions Destination: section.4 Item: Title: References Destination: section.4 Info: Title: Electrochemical Genosensing of E. coli Based on Padlock Probes and Rolling Circle Amplification Author: Alejandra Ben Aissa, Narayanan Madaboosi, Mats Nilsson and Maria Isabel Pividori Subject: Isothermal amplification techniques are emerging nowadays for the rapid and accurate detection of pathogenic bacteria in low resource settings, where many infectious diseases are endemic, and the lack of reliable power supply, trained personnel and specialized facilities pose critical barriers for timely diagnosis. This work addresses the detection of E. coli based on DNA isothermal amplification performed on magnetic particles (MPs) followed by electrochemical genosensing on disposable electrodes by square-wave voltammetry. In this approach, the bacterial DNA is preconcentrated using a target-specific magnetic probe and then amplified on the MPs by rolling circle amplification (RCA). Two different electrochemical readout methods for the RCA amplicons are tested. The first one relied on the labelling of the magnetic RCA product with a digoxigenin probe followed by the incubation with antiDIG-HRP antibody as electrochemical reporter. In the second case, the direct detection with an HRP-probe was performed. This latter strategy showed an improved analytical performance, while simultaneously avoiding the use of thermocyclers or bulky bench top equipment. 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