Olivier Y.F. Henry

Fabrication of molecularly imprinted polymer microarray on a chip by mid-infrared laser pulse initiated polymerisation

The possibility to assess several functional polymeric materials in parallel in a microchip format could find a wide range of applications in sensing, combinatorial and high-throughput screening. However several factors, inherent to the nature of material polymerisation have limited such development. We here report an innovative fabrication approach for the elaboration of polymer microarrays bearing polymer dots typically 300 microm in diameter fabricated in situ on a glass cover slip via CO(2) laser pulse initiated polymerisation, as well as initial results on the identification of a suitable monomer composition for the molecular imprinting of dansyl-L-phenylalanine as a proof-of-concept example. A combination of methacrylic acid and 2-vinylpyridine showed the largest affinity to dansyl-L-phenylalanine which agreed with the existing literature and the results were further confirmed by HPLC. Finally, a sensor chip bearing both non-imprinted as well as imprinted polymers was also prepared in order to prove the suitability of this fabrication approach for the elaboration of MIP based sensors. The assay consisted in a simple dip-and-read step and the sensing system was able to discriminate between the l and d enantiomers of dansylphenylalanine with an imprinting factor of 1.6.

Electrochemical detection of Francisella tularensis genomic DNA using solid-phase recombinase polymerase amplification

Solid-phase isothermal DNA amplification was performed exploiting the homology protein recombinase A (recA). The system was primarily tested on maleimide activated microtitre plates as a proof-of-concept and later translated to an electrochemical platform. In both cases, forward primer for Francisella tularensis holarctica genomic DNA was surface immobilised via a thiol or an amino moiety and then elongated during the recA mediated amplification, carried out in the presence of specific target sequence and reverse primers. The formation of the subsequent surface tethered amplicons was either colorimetrically or electrochemically monitored using a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the elongated strand. The amplification time was optimised to amplify even low amounts of DNA copies in less than an hour at a constant temperature of 37°C, achieving a limit of detection of 1.3×10(-13) M (4×10(6) copies in 50 μL) for the colorimetric assay and 3.3×10(-14) M (2×10(5) copies in 10 μL) for the chronoamperometric assay. The system was demonstrated to be highly specific with negligible cross-reactivity with non-complementary targets or primers.

Real-time and label-free ring-resonator monitoring of solid-phase recombinase polymerase amplification

In this work we present the use of a silicon-on-insulator (SOI) chip featuring an array of 64 optical ring resonators used as refractive index sensors for real-time and label-free DNA detection. Single ring functionalisation was achieved using a click reaction after precise nanolitre spotting of specific hexynyl-terminated DNA capture probes to link to an azido-silanised chip surface. To demonstrate detectability using the ring resonators and to optimise conditions for solid-phase amplification, hybridisation between short 25-mer single stranded DNA (ssDNA) fragments and a complementary capture probe immobilised on the surface of the ring resonators was carried out and detected through the shift in the resonant wavelength. Using the optimised conditions demonstrated via the solid-phase hybridisation, a 144-bp double stranded DNA (dsDNA) was then detected directly using recombinase and polymerase proteins through on-chip target amplification and solid-phase elongation of immobilised forward primers on specific rings, at a constant temperature of 37°C and in less than 60min, achieving a limit of detection of 7.8·10(-13)M (6·10(5) copies in 50µL). The use of an automatic liquid handler injection instrument connected to an integrated resealable chip interface (RCI) allowed programmable multiple injection protocols. Air plugs between different solutions were introduced to prevent intermixing and a proportional-integral-derivative (PID) temperature controller minimised temperature based drifts.

Electrode surface nanostructuring via nanoparticle electronucleation for signal enhancement in electrochemical genosensors

Biosensor read out signals can be enhanced by carefully designing the transducer surfaces to achieve an optimal interaction between the recognition elements immobilised and the targeted analyte. This is particularly evident in the case of genosensors, where spacing and orientation of immobilised DNA capture probes need to be controlled to maximise subsequent surface hybridisation with the target sequence and achieve high binding signals. Addressing this goal, we present a novel approach based on the surface nanostructuring of glassy carbon electrodes (GCEs) towards the development of highly sensitive electrochemical genosensors. Gold nanoparticles were sequentially electrochemically nucleated on glassy carbon electrodes to form dense arrays of randomly distributed gold nanodomains. The number density of the electronucleated nanoparticles could be increased by repeatedly alternating between a short electronucleation step and the subsequent insulation of the nucleated nanoparticles with thiolated DNA probes. This approach allowed for the creation of highly structured surfaces whilst preventing aggregation of nanoparticles. The performances of planar gold electrodes and that of the nanopatterned surfaces prepared following several rounds of deposition were compared for the amperometric detection of DNA. Three rounds of deposition exhibited the highest sensitivity (44.89 nA × nM(-1)), with a dynamic detection range spanning from 0.53 nM to 25 nM of the targeted sequence, i.e. one order of magnitude lower than that obtained for the planar gold electrodes. The use of the nanostructured surface we report here may find application not only in DNA biosensors but also for any sensing application requiring highly sensitive measurements.

Three-dimensional Arrangement of Short DNA Oligonucleotides at Surfaces via the Synthesis of DNA-branched Polyacrylamide Brushes by SI-ATRP

Short DNA oligonucleotide branches are incorporated into acrylamide brushes via surface initiated atom transfer radical polymerization in an attempt to increase DNA surface density by building three-dimensional molecular architectures. ATR-FTIR as well as hybridization studies followed by SPR confirm the incorporation of the DNA sequences into the polymer backbone. MALDI-TOF analysis further suggests that six acrylamide monomer units are typically separating DNA branches present on a single brushes approximately 26 units long. This new approach offers a promising alternative to SAM-based nucleic acid and aptamer sensors and could enable the realization of more complex soft materials of controlled architecture capable of both recognition and signaling by including additional optically or electrochemically active moieties.

Automated genotyping of circulating tumor cells

Cancer remains a prominent health concern in modern societies. Continuous innovations and introduction of new technologies are essential to level or reduce current healthcare spending. A diagnostic platform to detect circulating tumor cells (CTCs) in peripheral blood may be most promising in this respect. CTCs have been proposed as a minimally invasive, prognostic and predictive marker to reflect the biological characteristics of tumors and are implemented in an increasing number of clinical studies. Still, their detection remains a challenge as they may occur at concentrations below one single cell per ml of blood. To facilitate their detection, here we describe microfluidic modules to isolate and genotype CTCs directly from clinical blood samples. In a first cell isolation and detection module, the CTCs are immunomagnetically enriched, separated and counted. In a second module and after cell lysis, the mRNA is reversely transcripted to cDNA, followed by a multiplex ligation probe amplification of 20 specific genetic markers and two control fragments. Following the multiplex ligation probe amplification reaction, the amplified fragments are electrochemically detected in a third and final module. Besides the design of the modules, their functionality is described using control samples. Further testing using clinical samples and integration of all modules in a single, fully automated smart miniaturized system will enable minimal invasive testing for frequent detection and characterization of CTCs.

Electrochemical Genetic Profiling of Single Cancer Cells

Recent understandings in the development and spread of cancer have led to the realization of novel single cell analysis platforms focused on circulating tumor cells (CTCs). A simple, rapid, and inexpensive analytical platform capable of providing genetic information on these rare cells is highly desirable to support clinicians and researchers alike to either support the selection or adjustment of therapy or provide fundamental insights into cell function and cancer progression mechanisms. We report on the genetic profiling of single cancer cells, exploiting a combination of multiplex ligation-dependent probe amplification (MLPA) and electrochemical detection. Cells were isolated using laser capture and lysed, and the mRNA was extracted and transcribed into DNA. Seven markers were amplified by MLPA, which allows for the simultaneous amplification of multiple targets with a single primer pair, using MLPA probes containing unique barcode sequences. Capture probes complementary to each of these barcode sequences were immobilized on a printed circuit board (PCB) manufactured electrode array and exposed to single-stranded MLPA products and subsequently to a single stranded DNA reporter probe bearing a HRP molecule, followed by substrate addition and fast electrochemical pulse amperometric detection. We present a simple, rapid, flexible, and inexpensive approach for the simultaneous quantification of multiple breast cancer related mRNA markers, with single tumor cell sensitivity.

Development of MIP sensor for monitoring propofol in clinical procedures

Propofol is a widely used intravenous anaesthetic which requires close patient monitoring that involves blood extraction methods. Polymers with high affinity for propofol were computationally designed and their binding affinity tested. A diethylaminoethyl methacrylate based molecularly imprinted polymer was optimised in solid-phase extraction experiments showing no cross-reactivity to injections of urea, glucose, alfentanil and morphine. These polymers were initially integrated with screen-printed electrodes by photopolymerisation and used to detect propofol amperometrically, before modification to include conductive carbon to create better communication between the recognition sites of the polymer and the electrode. The sensors exhibited excellent linearity across the relevant clinical range with a detection limit of 4.19 µM measured in the presence of highly electroactive interferents such as uric acid and ascorbic acid. The mode of operation of the sensor reported herein differs significantly from the current state of the art and opens new opportunity towards the integration of synthetic receptors as electrochemical biosensors for point-of-care applications.