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On-chip isothermal nucleic acid amplification on flow-based chemiluminescence microarray analysis platform for the detection of viruses and bacteria Andreas Kunze, Mike Dilcher, Ahmed Abd El Wahed, Frank Hufert, Reinhard Niessner, and Michael Seidel Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03540 • Publication Date (Web): 01 Dec 2015 Downloaded from http://pubs.acs.org on December 2, 2015
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Analytical Chemistry
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On-chip isothermal nucleic acid amplification on flow-based
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chemiluminescence microarray analysis platform for the detection
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of viruses and bacteria
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Authors:
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A. Kunze1, M. Dilcher², A. Abd El Wahed³, F.Hufert4, R. Niessner1, M. Seidel1,*
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1
7
Munich, Germany
8
2
9
Kreuzbergring 57, D-37075 Göttingen, Germany
Institute for Hydrochemistry, Technical University Munich, Marchioninistr. 17, D-81377
Department of Virology, University Medical Center, Georg-August-University Göttingen,
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3
11
Burckhardtweg 2, D-37077 Göttingen, Germany
12
4
13
Grossenhainer Str. 57, D-01968 Senftenberg, Germany
Division of Microbiology and Animal Health, Georg-August-University Göttingen,
Institute of Microbiology and Virology, Brandenburg Medical School Theodor Fontane,
14 15
*Corresponding author: Email:
[email protected], Phone: +49-89-2180-78238, fax
16
+49-89-2180-78255
17 18
Abstract
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This work presents an on-chip isothermal nucleic acid amplification test (iNAAT) for the
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multiplex amplification and detection of viral and bacterial DNA by a flow-based
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chemiluminescence microarray. In a principle study, on-chip recombinase polymerase
22
amplification (RPA) on defined spots of a DNA microarray was used to spatially separate the
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amplification reaction of DNA from two viruses (Human adenovirus 41, Phi X 174) and the
24
bacterium Enterococcus faecalis, which are relevant for water hygiene. By establishing the
25
developed assay on the microarray analysis platform MCR 3, the automation of isothermal
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multiplex-amplification (39 °C, 40 min) and subsequent detection by chemiluminescence
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imaging was realized. Within 48 min the microbes could be identified by the spot position on 1 ACS Paragon Plus Environment
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the microarray while the generated chemiluminescence signal correlated with the amount of
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applied microbe DNA. The limit of detection (LOD) determined for HAdV 41, Phi X 174 and
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E faecalis was 35 GU/µL, 1 GU/µL and 5×10³ GU/µL (genomic units), which is comparable
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to the sensitivity reported for qPCR analysis, respectively. Moreover the simultaneous
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amplification and detection of DNA from all three microbes was possible. The presented
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assay shows that complex enzymatic reactions like an isothermal amplification can be
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performed in an easy-to-use experimental setup. Furthermore, iNAATs can be potent
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candidates for multi-pathogen detection in clinical, food or environmental samples in routine
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or field monitoring approaches.
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1. Introduction
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Nucleic acid amplification tests (NAATs) are powerful technologies in the field of
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clinical diagnostics1, food safety2, water safety3,
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because microbes like pathogenic bacteria and viruses are detected rapidly.6
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Cultivation-dependent microbiological methods will be replaced by NAATs in routine
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or field applications if cost-effective, user-friendly technologies are available and
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comparable results in each laboratory are achieved.7 Therefore, research on
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miniaturized NAATs is important to reduce costs for instrumentation, reagents, process
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times and manual process steps.8 Automated sample processing, nucleic acid
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amplification and detection have to be combined in a closed system to minimize
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contamination.9 Especially in the field of water safety, the development of such
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analysis platforms for the quantification of microbes would be a valuable task. The
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quantification of multiple pathogens is highly demanded because legally threshold
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levels for several pathogens and indicator organisms are defined from authorities.10
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Moreover, the range of biological parameters will be enlarged in the future to improve
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the water quality and potent detection methods for the monitoring of these parameters
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are needed.
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The most established nucleic acid amplification method is based on polymerase chain
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reaction (PCR). The quantification of microbes is either based on real-time PCR by
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fluorescence detection during thermocycling in homogeneous phase11 or by
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quantification of PCR products using heterogeneous hybridization assays on
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biosensors12, microarray chips13, lateral flow dipsticks14 or microbeads.15 Thereby,
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label-dependent (fluorescence16, chemiluminescence (CL)17, electrochemical18) or
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label-free19
(SPR,
RIFS,
etc.)
hybridization
4
and environmental monitoring5
assays
were
established.
The 2
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thermocycling in PCR requires sophisticated temperature control which results in
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expensive and complex PCR devices.20 Therefore, heating and cooling intervals have
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to be adapted for each PCR system. In consequence, PCR-based NAATs are not
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simply transferable between laboratories.
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Isothermal NAAT (iNAAT) is an emerging molecular-based analysis principle that
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overcomes the described restrictions of PCR and simplifies the quantification
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method.21 The amplification of nucleic acids takes place at constant temperature
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throughout the entire biochemical reaction.22 Besides a defined set of primers and
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polymerases, additional proteins are needed for the selective isothermal nucleic acid
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amplification reaction. A good overview of iNAATs preliminary developed for point-
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of-care testing was given by Craw and Balachandran.23 A part of iNAATs require an
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initial heating step to separate double stranded DNA, which complicates integration
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into (fluidic) microsystems. Examples are rolling circle amplification (RCA)24, strand
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displacement amplification (SDA)25 and isothermal and chimeric primer-initiated
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amplification of nucleic acids (ICAN).26 The initial heating steps of up to 95 °C used in
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these assays cause unwanted bubble formation in microsystems. Additionally, different
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reaction temperatures require more complex temperature control systems. Therefore,
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these isothermal NAAT technologies are challenging to integrate in flow-based
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microsystems and iNAATs using i.e. the strand-displacement activity of a protein are
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more favorable.20 On the other hand, nucleic acid sequence based amplification
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(NASBA) is only applicable to RNA molecules.27 As the detection of pathogens
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includes DNA and RNA coding viruses, NASBA is limited in its application for the
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multiplex analysis. Other technologies which use strand displacing proteins for
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separation of double stranded DNA are e.g. loop mediated isothermal amplification
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(LAMP)28, helicase dependent amplification (HDA)29 or recombinase polymerase
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amplification (RPA).30
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Many iNAATs were developed as miniaturized homogeneous assays using parallel
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nucleic acid amplification and fluorescence detection.31 Also heterogeneous iNAAT
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platforms were developed that mostly consist of physically separated chambers for
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homogeneous amplification and heterogeneous amplicon detection. For example, a
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multiplex iNAAT was developed by combination of NASBA with microarray
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analysis.32 For the detection of single pathogenic bacteria or viruses, lateral flow
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dipsticks were combined e.g. with LAMP33, SDA34,
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miniaturized RPA on a lab-on-disk (LoD) platform was performed by Lutz et al.38
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and RPA.36,
37
A first
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Later, Kim et al.39 integrated chambers for magnetic bead-based DNA extraction, RPA
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and amplicon quantification on lateral flow strips into one LoD instrumentation. Thus,
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Salmonella in milk was detected within 30 min. However, the combination of on-chip
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amplification and detection, multiplex analysis and assay automation is not yet solved.
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Regarding adaptability for different bioanalytical tasks microarray-based RPA seems
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to be most promising. RPA has the advantage that a relatively simple reaction scheme
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is applied by usage of two primers and two enzymes. Moreover, the amplification
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reaction is fast (40 to 60 min) and takes place at low reaction temperatures (37 °C to
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42 °C).30 Furthermore, only a set of two primers per target is needed (compared to a set
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of 6 primers for LAMP) and cDNA synthesis of RNA can be integrated.40 Microarray-
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based RPA is a promising technology for multiplex iNAATs, because separate
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positioned and immobilized primers will react independently with target DNA. No
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competition between enzymes and multiple substrates (nucleic acids) will occur.
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Several on-chip RPA methods have been reported. A solid-phase RPA on static
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incubated microarrays on a DVD was developed for the chromomeric detection of
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Salmonella.41 Similar limits of detection (LOD) were obtained with PCR and RPA for
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pathogens in skimmed milk samples by using a DVD-technology for detection of
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amplicons.42 In a multiplex on-chip RPA approach, Kersting et al. were able to detect
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three different pathogenic bacteria by a fluorescence microarray scanner after
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incubation of DNA microarrays in a hybridization chamber.43 However, the integration
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of on-chip amplification and detection in an automated analysis platform was not
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shown so far.
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The MCR 3 (microarray chip reader, third generation) has been used for the automated
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and rapid processing of flow-based CL microarrays in the field of food and water
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safety.44
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immunoassays.45 The MCR 3 was further developed after we have shown that rapid
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and sensitive hybridization assays can be performed on flow-based CL DNA
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microarrays.17 Therefore, the microarray chip loading unit was completely
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reconstructed to enable a defined temperature-control on the microarray surface and to
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integrate a hybridization chamber in the MCR 3.46 Single PCR-reactions were
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conducted for three viruses and pooled amplicons were analyzed in parallel with flow-
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based CL DNA microarrays. In this work, we show the first time that on-chip RPA and
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subsequent CL detection can be performed on the MCR 3 in an automated and
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multiplexed fashion.
The
instrument
was
preliminary
established
for
CL
microarray
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In a principle study the on-chip iNAAT was applied for HAdV 41, which is associated
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with gastroenteritis and diarrhoea in infants47 caused by contaminated water48,
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Subsequently, the feature of multiplexing was shown by detection of three water-
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related microbes (HAdV 41, Phi X 174 and E. faecalis).
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2. Experimental
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2.1 Materials, plasmid standards and primer design
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Standard chemicals, as well as casein, 2-amino-2-hydoxymethyl-propane-1,3-diol
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(TRIS),
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triethylamine and anhydrous dimethylformamide (DMF) were analytical grade and
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obtained from Sigma Aldrich (Taufkirchen, Germany). Streptavidin-horseradish
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peroxidase (strep-HRP) was purchased from Vector Laboratories (Burlingame, USA).
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Standard microscopic glass slides (76 x 26 x 1 mm) were obtained from Carl Roth
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(Karlsruhe, Germany). Chemiluminescence reagents (Westar Supernova ELISA
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luminol and hydrogen peroxide) were acquired from Cyanogen (Bologna, Italy).
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Oligonucleotide primers used in this study were synthesized by Eurofins Genomics
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(Ebersberg, Germany). RPA reactions were performed, using the TwistAmpTM basic
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kit from TwistDx (Babraham, UK). Primers were designed using the TwistDx
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instruction manual and the Seqbuilder modul of DNASTAR Lasergene Suite Version
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10 (DNASTAR, USA). Amplification curves of all possible primer combinations were
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compared in response a genome standard of 105 GU/µL. Those primer combinations
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showing the highest specificity and sensitivity were selected. The resulting primer
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sequences are summarized in Table 1.
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Table 1: Primers used in this study.
di(N-succinimidyl)-carbonate (DSC),
Target gene Human adenovirus 41
dimethylaminopyridine
Oligonucleotide sequence [5‘-3‘]
5'-Biotin-
CGTGGGTCGGAGCCACAGTGGGGTTTCTGAACTT
5'-NH2-C12
PhiX174-FW2
CAAAGTTTGGATTGCTACTGACCGCTCTCGTGCTC
5'-Biotin-
PhiX174-RW2
CGCCTTCCATGATGAGACAGGCCGTTTGAATG
5'-NH2-C12
Bacteriophage ΦX174
Enterococcus faecalis
Amplicon length 142 bp
GCCCCAGTGGTCATACATGCACATCGCCGGGCAGG
hAdV41-RW5
.
(DMAP),
Modification
Hexon
hAdV41-FW1
49
Protein D
181 bp
Superoxide dismutase sodA
Efaecalis-FW3
CAAACCATACATTCTTCTGGGAAATTATGGCACC
5'-Biotin-
Efaecalis-RW
CCAAAGCGGCCAGTTGCAGCTGTTTTGAAAG
5'-NH2-C12
144 bp
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For the generation of plasmid standards approximately 500 bp of the target genes were
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amplified using reference DNA of HAdV 41 (Sample code GastroV12-06, QCMD
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Viral Gastroenteritis Panel 2012), Bacteriophage Phi X 174 (#SD0031, Thermo
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Scientific, Germany) and Enterococcus faecalis (#DSM 20478, DSMZ, Braunschweig,
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Germany). The PCR products were cloned using the TA-cloning kit dual promoter
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with One shot® chemically competent E.coli (Invitrogen, Darmstadt, Germany) and
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the sequence was confirmed using Sanger sequencing (Seqlab, Goettingen, Germany).
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The recombinant pCRII plasmid was linearized with HindIII (FastDigest, Thermo
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Fisher Scientific) and the DNA concentration was quantified using Quant-iTTM Pico
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Green® dsDNA Assay Kit (Thermo Fisher Scientific). Based on the molecular weight
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of the linearized plasmid standard and the DNA concentration, the amount of genomic
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units per µL (GU / µL) was calculated. DNA plasmid standards were stored at – 20 °C
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until use.
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2.2 Microarray production
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Poly (propylene glycol) diamine (DAPPG) coated glass slides were produced in-house
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and used as solid support. The coating procedure was done as described in detail
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elsewhere.50,
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poly (ethylene glycol) diamine (DAPEG). DAPPG glass slides were activated using a
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mixture of 160 mg (0.62 mmol) of DSC and 8 mg (0.065 mmol) of DMAP dissolved in
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3.2 mL of absolute DMF and 250 µL of trimethylamine. Two activated glass slides
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were incubated at RT in a sandwich format with 600 µl of the prepared solution for
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4 h. After incubation, the slides were separated and sonicated (15 min) in methanol for
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cleaning. Finally, the slides were dried under nitrogen flow. Spotting was done by an
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unmodified piezo dispense capillary on the sciFLEXARRAYER S1 (Scienion AG,
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Germany) micro-drop dispensing system at 20 °C and 55% of humidity. Depending on
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the experiment, one to three modified primers as well as a spotting and a negative
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control were immobilized in five replicates, respectively. Thus, two 3 x 5 to 5 x 5
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(number of columns x number of lines) clusters per slide were generated. The distance
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between lines and columns were 1.1 mm and 1.3 mm, respectively. Spotting solutions
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were prepared in a polypropylene microtiterplate (sciSCOURCEPLATE-384-PP,
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Scienion AG, Germany). As spotting control, EZ-Link® amine-PEG2-biotin
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(0.005 mg/mL, Thermo Scientific, Germany) was used, whereas ultrapure water (PCR
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grade, Roche, Germany) served as negative control. In total, 28 ± 2 pmol of reverse
51
JEFFAMINE® ED-2003 (Huntsman, USA) was used instead of
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primer (with a 5’-amine-C12H24-modification) per target pathogen were spotted. After
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spotting, the glass slides were incubated over night at 60 °C and 55% of humidity,
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followed by a blocking step with TRIS-HCl (1 M TRIS, pH 8.5) for 15 min and
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subsequent washing with water (MilliQ) and methanol for 1 min. After drying under
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nitrogen flow, the glass slides could be stored in the dark at – 20 °C for several weeks.
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Prior to the experiment, the microarray was formed by the prepared glass slide and a
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(poly) methyl methacrylate (PMMA) solid support via a double-sided adhesive foil
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(Adhesives Research, Ireland) as published elsewhere.45 One microarray chip
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contained two flow cells and therefore was used for two experiments.
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2.3 Microarray analysis platform MCR 3
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The MCR 3 was developed at our institute over the last years44 and build by GWK
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Präzisionstechnik GmbH (Munich, Germany) as a stand-alone microarray analysis
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platform for the automated processing of flow-based chemiluminescence microarrays.
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The readout is performed by chemiluminescence imaging after enzymatic reaction of
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HRP with luminol and H2O2 on each spot of the microarray.45 Due to the
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reconstruction of the chip loading unit and the upgrade by a thermoelectric heating
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module as reported previously46, the temperature on the microarray surface can now be
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controlled with a precision of ± 1 °C. Thus, the integration of an isothermal
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amplification into the MCR 3 was possible. In order to simplify the fluidic setup and
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prevent cross contamination, the procedure for sample injection was adapted. The
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sample was injected directly into the flow cell of the microarray chip (Figure 1 A) and
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the flow of reagents was designed to be one-way (Figure 1 B). Thus, the contact
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between sample and fluidic system was minimized and the number of steps in the
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sequence program could be cut down. Consequently, only a minimum of fluidic
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components was necessary to perform the assay (theoretically, one valve and one
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syringe pump) and the time for incubation with reagents as well as for rinsing of the
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fluidic setup between the experiments could be reduced.
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Figure 1: (A) Work-flow for the sample preparation and injection into the microarray chip. (B) Flow chart of the on-chip RPA on the MCR 3. During on-chip RPA (step 1) biotinylated amplification products are bound on the microarray surface. After washing, biotin is detected by strep-HRP (step 2) and finally HRP catalyzes a CL reaction which is recorded by a CCD camera (step 3).
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2.4 On-chip isothermal amplification and automated detection
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The running buffer for on-chip RPA experiments was casein (0.5 % (w/w)) in
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phosphate-buffered saline (PBS, 145 mM NaCl, 10 mM KH2PO4 and 70 mM K2HPO4,
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adjusted to pH 7.6). Strep-HRP solution was prepared in running buffer (0.4 µg/mL).
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Prior to the experiments, running buffer, chemiluminescence substrates and strep-HRP
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were degassed by sonication for 20 min. Tubes, valves and syringe pumps of the
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fluidic system were washed intensively and filled with reagents. For each disposable
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microarray chip, the background signal of the CCD camera was recorded for 60 sec
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and automatically subtracted from the measurement picture. The measurement
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program was started and the flow cell was equilibrated at the aspired temperature.
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Using a specially designed pipetting adapter, 52 µL of the freshly prepared reaction
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mix was transferred to the microarray (Figure 1 A). If not stated otherwise, a typical
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RPA reaction (singleplex, total volume of 54 µL) contained 0.93 µM of biotinylated
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forward primer, 0.093 µM of unmodified reverse primer, 10.5 µL of water (PCR
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grade) and 29.5 µL of 1 x Twist Dx rehydration buffer. After rehydrating the freeze-
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dried reaction pellet with 45 µL of the master mix, 5 µL of DNA template were added.
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Finally, the reaction was initiated with 20.74 mM of magnesium acetate. The loading
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of the microarray chip was followed by the incubation / amplification step at constant,
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defined temperature and time (Figure 1 B, step 1). After amplification, the flow cell 8 ACS Paragon Plus Environment
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was washed three times with 1 mL (200 µL/s) of running buffer at 37 °C. Biotinylated
241
amplification products bound during amplification were detected in the next step by
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strep-HRP (600 µL, 2 µL/s, 37 °C, Figure 1 B, step 2). Unbound strep-HRP was
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removed by rinsing the microarray chip three times with 1 mL (200 µL/s) of running
244
buffer at 37 °C. Finally, in total 400 µL of the chemiluminescence reagents luminol
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and H2O2 premixed in a syringe (1:1, (v/v)), were pumped through the microarray chip
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with 20 µL/s at 35 °C. The chemiluminescence signal was recorded by the CCD
247
camera for 60 s, while liquid flow was stopped (Figure 1 B, step 3). The complete
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fluidic system was rinsed three times with running buffer after every experiment, to
249
prevent cross-contamination. After the insertion of the microarray chip into the chip
250
loading unit, all program steps are performed automatically and the measurement
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image can directly be analyzed on the MCR 3.
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2.5 Dose-response curves
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To gain dose-response curves for HAdV 41, Phi X 174 and E. faecalis, linearized
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plasmid standard DNA was applied in concentrations of 0 to 1 x 107 GU/µL. The
255
dilutions were prepared in tRNA solution (from yeast, 100 µg/mL, Sigma Aldrich,
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Taufkirchen, Germany) to increase the stability of the DNA standards. A logistic
257
regression was applied to fit the correlation between CL signal and log of the applied
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DNA concentration. The detection limit was calculated according to IUPAC as
259
തݔതതത + 3ܵ gained from three blank measurements.52 The resulting CL value was applied
260
to the regression equation and the corresponding concentration (LOD) was calculated.
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2.6 Read-out and data-analysis
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Every experiment on the MCR 3 resulted in a 2 D chemiluminescence image (2 x 2
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binning mode, 696 x 520 pixels), which was analyzed by MCRVisualization 1.0.6
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(GWK Präzisionstechnik, Germany). The chemiluminescence intensity is described as
265
grey level intensity per pixel and ranged from 0 to 65536 a.u. (16 bit CCD camera). A
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blank picture was subtracted from the measurement picture for data evaluation. A grid
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was applied to the spots and a mean value was calculated from the 10 brightest pixels
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per spot. The resulting values of the five replicates per immobilized target were
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averaged.53 If the value of one spot exceeded a deviation of 15% compared to the mean
270
value of the five replicates, it was marked as an outlier. The results were exported as
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primers SCLpathogen were normalized by the chemiluminescence value for maximum
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saturation of the CCD camera SCLmax. SCLmax was calculated individually for every
274
microarray chip by subtraction of the averaged intensity of the blank picture from the
275
maximum grey level intensity of the CCD camera (65536 a.u.).
276
3. Results and Discussion
277
3.1 Automated, asymmetric on-chip RPA
278
Several on-chip RPA methods have been reported but are not yet suitable to fulfill the
279
stated requirements for routine measurements (multiplex pathogen detection, easy-to-
280
use experimental setup and protocols, prevention of cross-contamination) because the
281
automation level is limited. Moreover, both symmetric and asymmetric primer
282
conditions were used. Del Rio et al.54 and Shin et al.55 applied only the non-
283
immobilized primer to the reaction mix. Hence the amplification only took place on the
284
solid phase (Figure 2, right). By applying both primers to the reaction mix, Kersting
285
et al.43 aimed to trigger a second amplification cycle in the bulk phase above the
286
microarray surface (Figure 2, left). Consequently, amplicons produced in the bulk
287
phase would hybridize to the immobilized primer on the surface thereby enhancing the
288
sensitivity of the assay.42 We have integrated an on-chip asymmetric RPA-based
289
amplification and a subsequent detection into our stand-alone microarray analysis
290
platform MCR 3. Thus, for the first time an on-chip RPA was established on a flow-
291
based chemiluminescence DNA microarray. A scheme of the assay principle is shown
292
in Figure 2. The prepared reaction mix, including DNA sample is injected into the flow
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cell of the microarray chip which is inserted into the chip loading unit (Figure 1 A &
294
B). During amplification, unlabeled reverse primer (immobilized on the microarray
295
surface and in the bulk phase) as well as biotin-labeled forward primer forms a
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complex with a recombinase. The recombinase / primer complex scans the template
297
DNA for homologous sequences and invades the DNA double strand. The displaced
298
strand is bound by single strand DNA binding proteins and primers are extended by the
299
polymerase. Biotinylated amplification products are immobilized during amplification
300
either by hybridization of labeled strands amplified in solution (Figure 2, left), or by
301
on-chip synthesis (Figure 2, right). Consequently, an immobilization of biotin occurs
302
only after successful amplification. Thereby, the reaction compartment is incubated
303
statically during amplification. A dynamic incubation (movement of the compartment
304
during the incubation) was tested, but had negative influence on amplification 10 ACS Paragon Plus Environment
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efficiency (data not shown). After incubation, unbound nucleic acids and amplification
306
reagents are removed by a washing step. A contamination of the fluidic system thereby
307
is prevented as the flow of reagents is one-way. For the detection of immobilized
308
biotin, the strong affinity to streptavidin is used. Horseradish peroxidase labeled
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streptavidin binds to biotin and thereby enables chemiluminescence imaging. By using
310
chemiluminescence for detection, the sensitivity can easily be influenced by the
311
exposure time and the experimental setup can be simplified. The immobilization of
312
pathogen-specific primers on different spots on the microarray surface spatially
313
separates the amplification reaction. Since the amplification takes place individually
314
for one target pathogen per spot, a multiplexing of the developed assay is enabled. The
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pathogen is identified by the spot position on the microarray, while the
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chemiluminescence intensity correlates with the amount of pathogen DNA in the
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sample.
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319 320 321 322 323 324 325 326
Figure 2: Schematic of the assay principle for automated on-chip RPA: Unlabeled reverse primer (immobilized on the microarray surface and in the bulk phase) as well as biotinlabeled forward primer forms a complex with a recombinase (1). The recombinase/primer complex scans the template DNA for homologous sequences and invades the DNA double strand (2). The displaced strand is bound by single strand DNA binding proteins (3) and primers are extended by a polymerase (4). Biotin is immobilized either by hybridization of labeled strands amplified in solution or on-chip synthesis. 11 ACS Paragon Plus Environment
Analytical Chemistry
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327 328
3.2 Optimization of reaction conditions
329
Enzymatic reactions like nucleic acid amplification are strongly influenced by
330
temperature in terms of efficiency, reproducibility and selectivity. Therefore, different
331
reaction temperatures of 20, 30, 37, 42, 50 and 60 °C were tested. The applied amount
332
of HAdV 41 DNA template was 106 GU/µL. The incubation time was 20 min in each
333
case. The results are shown in Figure 3 A. At temperatures from 20 to 30 °C the CL
334
signal was in the range of the background signal. For a temperature of 37 °C, the
335
background was exceeded to a normalized CL signal of 32 ± 8%. By increasing the
336
reaction temperature to 42 °C, the normalized CL signal was more than doubled
337
(72 ± 15%). The loss of reproducibility for a temperature of 42 °C was caused by a
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higher standard deviation of the background signal. This was based on unspecific
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binding of strep-HRP and biotinylated nucleic acids onto the microarray surface at
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higher incubation temperatures. At 50 °C, the standard deviation was further increased
341
which supported this assumption (50 ± 27% of maximum possible intensity). At
342
temperatures above 50 °C, the signal fell to the level of the background signal. It is
343
most likely that a denaturation of the proteins needed for amplification was responsible
344
for this signal decrease. Besides temperature, another fundamental parameter for
345
enzymatic reactions is reaction time. We tested the influence of reaction times of 5, 10,
346
20, 30, 40, 60, 120, 240 and 840 min at a constant temperature of 37 °C. A
347
concentration of HAdV 41 template of 104 GU/µL was chosen, representing a
348
concentration in the range of the detection limit. The results are shown in Figure 3 B.
349
Already a reaction time of 10 min resulted in a normalized CL signal of 4.3 ± 1.8%
350
above background. Further extension of the reaction time increased the assay
351
sensitivity by a factor of up to ~ 20 for a reaction time of 60 min (81 ± 8% of
352
maximum possible intensity). Thereby, the immobilization of biotin during the
353
amplification was relatively stable. After 14 h reaction time the normalized CL signal
354
was at 53 ± 19%. Independently from the parameter influencing the amplification
355
(reaction temperature and time), the maximum chemiluminescence value for HAdV 41
356
was at ~ 80% of the maximum possible intensity SCLmax (100%, saturation of the CCD
357
camera). Moreover, an optimization of the amplification efficiency could be reached
358
by reaction temperature or/and time. Rising reaction temperature led to an increase of
359
the CL signal, whereas the reproducibility was reduced through unspecific binding.
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The reaction time could be raised up to 60 min without negative influence on both 12 ACS Paragon Plus Environment
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361
background signal and reproducibility. Hence, a temperature of 39 °C was chosen for
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the optimized protocol, whereby the reaction time could be reduced from 60 to 40 min.
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Consequently, the overall measurement time of the developed assay was optimized to
364
48 min.
365 A
m = 3, n = 6
B
m = 3, n = 8
100
100
80
80
SCLHAdV 41/SCLmax / %
SCLHAdV 41/SCLmax / %
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60
40
20
0
60
40
20
0 20
30
40
50
60
0
20
Temperature / °C
40
60
80
100 120 140 160
800
1000
Incubation time / min
366 367 368 369 370 371 372
Figure 3: Optimization of reaction conditions for HAdV 41. (A) Signal derived from the HAdV 41 reverse primer immobilized on the microarray surface for different reaction temperatures. The reaction time remained constant at 20 min. (B) Signal derived from the HAdV 41 reverse primer immobilized on the microarray surface for different reaction times. The reaction temperature remained constant at 37 °C. “m” means number of replicates, “n” means number of measurement points.
373 374
3.3 Quantification
375
The optimized conditions for on-chip RPA (40 min, 39 °C) were used to determine
376
dose-response curves for HAdV 41, Phi X 174 and E. faecalis in a singleplex
377
experiment. The DNA plasmid standard was applied in concentrations of 0 to
378
107 GU/µL. The results are shown in Figure 4. For all three microbes a sigmoidal
379
correlation between the log of the applied DNA concentration and normalized CL
380
signal SCLpathogen was observed. The resulting limits of detection (LOD) were
381
35 GU/µL, 1 GU/µL and 5×10³ GU/µL for HAdV 41, Phi X 174 and E. faecalis,
382
respectively. Other recent studies are using solid-phase RPA combined with
383
electrochemical or label-free detection instead of chemiluminescence imaging. Del Rio
384
et al.54 used chronoamperometry to measure the oxidation of precipitated 3,3',5,5'-
385
tetramethylbiphenyl-4,4'-diamine (TMB) for detection. The reported LOD was
386
104 GU/µL, which is several orders of magnitude above the LOD of the presented 13 ACS Paragon Plus Environment
Analytical Chemistry
387
assay. On the other hand, Shin et al.55 applied a silicon microring resonator for
388
detection and were able to measure 500 fg/µL DNA, while our assay on the MCR 3
389
achieved < 0.5 fg/µL. Compared to qPCR analysis for HAdV 41, the sensitivity of the
390
presented assay was in the same range. For example, Heim et al.56 achieved a LOD of
391
15 copies/µL with TaqMan-qPCR.
392 120 HAdV 41 (m = 3, n = 9, R² = 0.989) Phi X 174 (m = 3, n = 7, R² = 0.998) E. faecalis (m = 3, n = 7, R² = 0.989)
100
80
SCLpathogen/SCLmax / %
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60
40
20
0 0 -3 10
100
101
102
103
104
105
106
107
108
DNA concentration / GUµL-1
393 394 395 396
Figure 4: Calibration curves for HAdV 41, Phi X 174 and E. faecalis at optimized conditions. A sigmoidal correlation between the log of the applied DNA concentration and the CL signal was observed. “m” means number of replicates, “n” means number of measurement points.
397 398
3.4 Multiplex RPA for waterborne pathogens
399
In order to establish iNAATs for applications in the area of environmental monitoring
400
and water safety, the parallel detection and identification of several bacteria and
401
viruses at the same time is preferred. As a proof-of-concept, three water relevant
402
microbes (HAdV 41, E. faecalis, and bacteriophage Phi X 174) were analyzed in
403
parallel in a composite sample. The optimized primer concentrations in the reaction
404
mix for multiplex on-chip RPA were 0.042 µM and 0.42 µM (reverse primer and
405
biotinylated forward primer) for E. faecalis and HAdV 41, respectively. For
406
bacteriophage Phi X 174 a primer concentration of 0.03 µM and 0.3 µM (reverse
407
primer and biotinylated forward primer) was used. To allow a comparison of the
408
generated CL signals the template DNA for each microbe was applied at the same
409
concentration (106 GU/µL). When DNA of only one microbe was used, a normalized
410
CL signal of 25 ± 2%, 4 ± 0.4% and 37 ± 4% was obtained for HAdV 41, E. faecalis
411
and Phi X 174, respectively. No cross reactivity signals were observed (data not 14 ACS Paragon Plus Environment
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412
shown). The results for duplex and triplex experiments are presented in Figure 5. When
413
HAdV 41 was applied with Phi X 174, normalized CL signals of 20 ± 0.9% and
414
35 ± 0.02% were generated while the normalized CL signal for E. faecalis was
415
0.1 ± 0.4%. For the combination of HAdV 41 and E. faecalis normalized CL signals of
416
19 ± 2% and 2 ± 0.3% were observed, whereas the Phi X 174 signal was 0.1 ± 0.2%.
417
Finally, the parallel detection of E. faecalis and Phi X 174 resulted in normalized CL
418
signals of 4 ± 1% and 39 ± 4%. The normalized CL signal for HAdV 41 was
419
0.8 ± 0.4%. Consequently, all duplex samples could be identified correctly. The
420
normalized CL signals for single and duplex experiments were comparable. A
421
composite sample containing all three microbes showed CL signals in a range similar
422
to single and duplex experiment. The normalized CL signals for HAdV 41, E. faecalis
423
and Phi X 174 were 36 ± 6%, 6 ± 1% and 44 ± 4%, respectively. Finally, the assay was
424
successfully applied for the simultaneous detection of genomic DNA from cultures of
425
Phi X 174 (produced in E. coli DSM 13127) and E. faecalis after DNA extraction by
426
chaotropic salts without further purification (Figure S-1). The results show, that the
427
developed on-chip RPA assay could be applied successfully for multiplex detection.
428
Moreover, the spatial separation of the reverse primers of different target organisms on
429
the microarray enabled a multiplex quantification.
430
431 432 433 434 435
Figure 5: Chemiluminescence images for multiplexing experiments. The applied amount of 6 target DNA was 10 GU/µL. The microarray contains a spotting control (SC) and a no target control (NTC, only spotting buffer). The amplification was carried out for 40 min at 39 °C.
436
4. Conclusion
437
We have shown for the first time that flow-based CL DNA microarrays can be used for
438
iNAAT suitable for the detection of viruses and bacteria at the same time. The on-chip
439
iNAAT using recombinase polymerase amplification on the microarray analysis 15 ACS Paragon Plus Environment
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Page 16 of 18
440
platform MCR 3 was established for quantitative detection of three drinking water
441
hygiene relevant microbes (HAdV 41, Phi X 174, E. faecalis). The sensitivity of the
442
developed assay was comparable to qPCR, while the measurement time was much
443
shorter. The complex enzymatic amplification reaction could be performed
444
individually for every microbe on spatially separated spots on the DNA microarray
445
chip by immobilization of microbe-specific primers. Thus, a multiplexing of the
446
amplification was achieved. The feature of multiplex on-chip iNAAT was
447
demonstrated successfully with composite samples containing plasmid standard DNA
448
of one, two or all three target microbes. The multiplex detection of genomic DNA from
449
cultures extracts was also possible. In further studies we want to further develop the
450
presented DNA microarray for hygiene monitoring of raw and drinking water.
451
Therefore, several indicator organisms (E. coli, E. faecalis, P. aeruginosa, MS 2,
452
Phi X 174) and pathogenic viruses and bacteria (i.e. HAdV 40, 41, rotaviruses,
453
enteroviruses, noroviruses, L. pneumophila, C. jejuni, K. pneumophila) have to be
454
implemented on a water hygiene chip system. Furthermore, a more detailed study
455
about cross-reactivities and sensitivities has to be conducted with a huge number of
456
water samples for acceptance by the authorities and end-users. Therefore, the nucleic
457
acid extraction and the preparation of the reaction mix for on-chip RPA have to be
458
implemented in the MCR 3 as next step. Thus, we will be able to fulfill the
459
recommendation of miniaturized analysis platforms for iNAATs as easy-to-use
460
portable systems for the detection of pathogens.22 In combination with appropriate
461
systems for the concentration of pathogens in water samples prior to the detection, like
462
i.e. ultrafiltration or monolithic filtration and automated systems for nucleic acid
463
extraction, a routine hygiene monitoring of large-volume drinking water samples
464
should be established.57 On-chip iNAATs in combination with flow-based CL DNA
465
microarrays have a great potential for the application in water hygiene and other
466
research fields like environmental monitoring, food safety and clinical diagnostics.
467 468
Acknowledgement
469
The authors like to thank the BMBF for financial support (project EDIT, 033W010E).
470
Especially we want to thank GWK Präzisionstechnik GmbH for their collaboration in
471
the project and the supply of the MCR 3 research device.
472
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