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RECOMBINASE POLYMERASE AMPLIFICATION IN RING
RESONATORS FOR REAL-TIME AND LABEL-FREE DETECTION OF dsDNA
Jonathan Sabaté del Ríoa,b, Tim Steylaertsa, Olivier Y. F. Henryc, Peter
Bienstmand, Tim Stakenborga, Wim Van Roya, Ciara K. O’Sullivanb,e*
Introduction
Methodology
Conclusion
This poster partly describes work undertaken in the context of EC FP7-ICT project 257743 "Magnetic Isolation and moleculaR Analysis of single CircuLating and disseminated tumor cElls on
chip (MIRACLE)” http://www.miracle-fp7.eu. The project is partially funded by the European Commission. It does not necessarily reflect its views and in no way anticipates the Commission’s future
policy in this area.
Acknowledgements:
Results
The surface of the chip is activated by the formation of an homogeneous self-
assembled monolayer of azidosilane achieved by vapor phase deposition. The
rings are then functionalised via a fast “click” reaction using a spotter for
functionalisation of specific ring resonators with hexynyl terminated DNA
sequences (either forward primers for RPA or complementary strands for direct
detection).
SURFACE ACTIVATION AND FUNCTIONALISATION MICROFLUIDIC ASSEMBLY
The hydrophobicity of the chip surface is
enhanced after the self assembled
monolayer of azidosilane is deposited,
increasing the contact angle from 73º to
90º.
1 2
SURFACE WETTABILITY1 SURFACE CHEMISTRY OPTIMISATION 2
The DNA probe density, spacer length and the backfiller-to-
probe ratio was optimised to enhance hybridization efficiency
The calibration curve of the ring resonators for the direct detection of ssDNA span five orders of magnitude, with a limit of detection (LOD) of 20
nM. Recombinase polymerase proteins were used in order amplify/detect dsDNA in solid phase by amplification of immobilised forward primers
on the chip at constant temperature, yielding an LOD of 7.8·10-13 M.
3 DIRECT DETECTION AND ENZYMATIC DNA AMPLIFICATION CALIBRATION CURVE4
Programmable robotic arm
with a nozzle dispenser
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log [DNA] / log M
LOD: 20 nM
Isothermal solid-phase amplification and detection of genetic markers can be performed without labelling in real-time by utilising both silicon microring resonators and solid-phase recombinase polymerase amplification. The technique was performed on a
silicon microring resonator array chip with a microfluidic chamber in contact to a temperature controller. For the solid-phase amplification, a hexynyl-terminated primer of the target was directly attached to the surface of each ring resonator with a spotting device
via click chemistry reaction. The amplified DNA was detected for each microring resonator by measuring the relative shift of the resonant wavelength during the DNA amplification on solid-phase. The probe spacing and surface density was optimised with
different back-filler ratios to reach the best hybridisation conditions on solid-phase. Assay time lasted less than an hour at a constant temperature of 37 ºC, with high sensitivity and selectivity, avoiding the primer engineering associated to liquid-phase RPA.
Silanised 90 ºCSiO2 73º
The resonant light circulating along the ring generates an evanescent field
several nanometers into the surrounding medium and interacts repeatedly
with the analytes near the surface. This interaction produces a change in the
refractive index, and therefore the resonance wavelength observed by the
camera shifts according to the amount of analyte interacting with thereceptors immobilised on the rings resonators
Solid-phase recombinase polymerase amplificationRing resonator detection mechanism
The use of microring resonators allowed the direct and labeless detection of ssDNA with with LOD as low as 20 nM. We have also demonstrated isothermal solid-phase recombinase polymerase amplification and detection of a low number of DNA copies
(105 copies/µL). Furthermore, the solid-phase approach overcomes the limitations present in regular RPA-based analysis, where the production of by-product DNA sequences can hinder the final analysis and, thus, requires the need for specific THF-based
engineered primers. Therefore, traditional PCR primers can be used in the developed approach highlighting the simplicity and genericity of the system reported here.
Forward
primer
Reverse
primer
Recombinase
protein
Binding
proteinsPolymerase
(1) (2) (3) (4)Ring resonator
Drop waveguide
Bus waveguide
Grating input
couplers
Coupling
region
Grating output
couplers
IR camera
IR laser
Receptor
Analyte
Evanescent
field
Ressonant
light
Inte
nsity
Wavelenght
Ring resonator chip
(1)Recombinase proteins form a complex with forward and reverse primers
(2)scan dsDNA for cognate sites
(3) introduce the primers in the template by a strand-displacement mechanism.
(4)The polymerase initiates primer elongation at their 3’ ends and exponential
amplification is achieved by cycling of this process
Time
Ring resonator array chip overview. Light source is collected at the grating input
couplers, directed through the waveguides towards the ring resonators array (8
columns of 4 pairs) and finally the resonant wavelength shift measured at the
grating output couplers
Top view of the re-sealable chip interface. The PMMA gasket has (1) two holes for the laser input
and the output reading, (2) five alignment pins to fix the chip in position, (3) four channels patterned
inside the PMMA and connected to (4) PDMS channels in contact with the chip, each one feeding 8
pairs of rings. (5) Ring resonator chip.
Illustration of the re-sealable chip interface disassembled in a (1) top
metallic clamp, (2) PMMA gasket patterned with a 4-channel PDMS
microfluidic unit, (3) aluminium seat for heat transfer to the chip, (4)
ring resonator chip, (5) metallic holder, (6) connectors and tubing.
Ring functionalisation
by click chemistry
Wavelength
shift
r1: 1.197 nm/min
500 nM
r2: 0.404 nm/min
250 nM
r3: 0.180 nm/min
125 nM
r4: 0.090 nm/min
60 nM 30 nM
r5: 0.045 nm/min ru: 0.012 nm/min
15 nM
r6: 0.022 nm/min
7 nM
r7: 0.011 nm/min
unspecific …
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Control ring resonator
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LOD: 7.8·10-13M
10-9 M
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a IMEC, Smart Systems and Emerging Technologies unit, Department of Life Science Technologies, Kapeldreef 75, 3001 Leuven, Belgium.b Nanobiotechnology and Bioanalysis Group, Departament d'Enginyeria Química, Universitat Rovira i Virgili, 26 Països Catalans, 43007 Tarragona, Spain.
c Currently located at The Wyss Institute for Biologically Inspired Engineering at Harvard University, 3 Blackfan Circle, Floor 5, Boston, MA 02115, United States.d IMEC - Ghent University, Photonics Research Group, Sint-Pieters-nieuwstraat 41, 9000 Ghent, Belgium.
e Institució Catalana de Recerca i Estudis Avançats, Passeig Lluís Companys, 23, 08010 Barcelona, Spain.
