Multiplex PCB-based electrochemical detection of cancer biomarkers using MLPA-barcode approach.

Asymmetric multiplex ligation-dependent probe amplification (MLPA) was developed for the amplification of seven breast cancer related mRNA markers and the MLPA products were electrochemically detected via hybridization. Seven breast cancer genetic markers were amplified by means of the MLPA reaction, which allows for multiplex amplification of multiple targets with a single primer pair. Novel synthetic MLPA probes were designed to include a unique barcode sequence in each amplified gene. Capture probes complementary to each of the barcode sequences were immobilized on each electrode of a low-cost electrode microarray manufactured on standard printed circuit board (PCB) substrates. The functionalised electrodes were exposed to the single-stranded MLPA products and following hybridization, a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the amplified strand completed the genocomplex, which was electrochemically detected following substrate addition. The electrode arrays fabricated using PCB technology exhibited an excellent electrochemical performance, equivalent to planar photolithographically-fabricated gold electrodes, but at a vastly reduced cost (>50 times lower per array). The optimised system was demonstrated to be highly specific with negligible cross-reactivity allowing the simultaneous detection of the seven mRNA markers, with limits of detections as low as 25pM. This approach provides a novel strategy for the genetic profiling of tumour cells via integrated "amplification-to-detection".

Fabrication and functionalization of PCB gold electrodes suitable for DNA-based electrochemical sensing.

The request of high specificity and selectivity sensors suitable for mass production is a constant demand in medical research. For applications in point-of-care diagnostics and therapy, there is a high demand for low cost and rapid sensing platforms. This paper describes the fabrication and functionalization of gold electrodes arrays for the detection of deoxyribonucleic acid (DNA) in printed circuit board (PCB) technology. The process can be implemented to produce efficiently a large number of biosensors. We report an electrolytic plating procedure to fabricate low-density gold microarrays on PCB suitable for electrochemical DNA detection in research fields such as cancer diagnostics or pharmacogenetics, where biosensors are usually targeted to detect a small number of genes. PCB technology allows producing high precision, fast and low cost microelectrodes. The surface of the microarray is functionalized with self-assembled monolayers of mercaptoundodecanoic acid or thiolated DNA. The PCB microarray is tested by cyclic voltammetry in presence of 5 mM of the redox probe K3Fe(CN6) in 0.1 M KCl. The voltammograms prove the correct immobilization of both the alkanethiol systems. The sensor is tested for detecting relevant markers for breast cancer. Results for 5 nM of the target TACSTD1 against the complementary TACSTD1 and non-complementary GRP, MYC, SCGB2A1, SCGB2A2, TOP2A probes show a remarkable detection limit of 0.05 nM and a high specificity.

Bipodal PEGylated alkanethiol for the enhanced electrochemical detection of genetic markers involved in breast cancer.

Extensive research efforts continue to be invested in the development of low-density electrochemical DNA sensor arrays for application in theranostics and pharmacogenomics. Rapid and low-cost technologies are thus required for genosensor arrays to impact on current medical practice, with sensors clearly being required to detect their targets with high sensitivity and specificity, whilst resisting biofouling and avoiding interfering signals from the sample matrix. We report on the performance of three polyethylene glycol (PEG) co-immobilisation strategies used in the preparation of DNA sensors, using the detection of the breast cancer marker oestrogen receptor-α as a model system. PEGylated DNA capture probes for oestrogen receptor-α were co-immobilised in the presence of either a PEG alkanethiol, a mixture of PEG alkanethiol and mercaptohexanol or a bipodal aromatic PEG alkanethiol. Electrochemical impedance spectroscopy and pulsed amperometry were employed to characterise the prepared surface and sensitivity of the sensor. A surface plasmon resonance study was additionally carried out to confirm the results obtained electrochemically. Finally, the best co-immobilisation system, consisting of the co-assembly of oestrogen receptor-α capture probes and bipodal aromatic PEG alkanethiol in a ratio of 1:100, was used for the electrochemical analysis of a PCR product resulting from the amplification of the genetic material extracted from 20 MCF7 cells. This novel co-immobilisation system exhibited both high electrochemical sensitivity and resistance to fouling believed to results from an enhanced electron permeability and surface hydrophilicity.

Electrochemcial characterisation and hybridisation efficiency of co-assembled monolayers of PEGylated ssDNA and mercaptohexanol on planar gold electrodes.

The realisation of efficient genosensors relies crucially on the surface chemistry strategy selected for the immobilisation of DNA probes. We report on the performance of surfaces prepared via the direct co-immobilisation of a 20-nucleotide (nt) thiolated single stranded DNA probe (ssDNA) in the presence of the short alkanethiol mercaptohexanol (MCH) at varying ratio. Electrochemical impedance spectroscopy was used for the physical characterisation of the formed monolayers. Functional characterisation of the surfaces was achieved by means of differential pulse voltammetry and calibration curves in the range 6.25-100 nM of the complementary sequence 104 nt in length were obtained for each surface studied, using the guanine specific redox marker methylene blue (MB) as hybridisation indicator. The best sensor performance was obtained for a ratio of 1:100, as previously suggested by electrochemical impedance spectroscopy. No hybridisation could be measured at 1:1 ratio and significantly lower hybridisation were recorded for surfaces prepared at 1:10 and 1:1000 ratio. For comparison, sensors prepared via initial thiolated ssDNA immobilisation followed by the backfilling of the surface with MCH, as widely reported, were also assessed. Our results suggested that the performances of such sensors were sub-optimum and comparable to that obtained at 1:10 co-immobilisation. We concluded that the co-immobilisation approach offers several advantages, with highly reliable surfaces being prepared in a single step.

Electrochemical quantification of DNA amplicons via the detection of non-hybridised guanine bases on low-density electrode arrays.

A new strategy for the electrochemical detection and signal amplification of DNA at gold electrodes is described. Current methodologies for DNA biosensing based on the electrochemical detection of electroactive base-specific labels such as methylene blue (MB) suffer from lengthy incubation and washing steps. Addressing these limitations, we report a novel approach for the electrochemical quantification of surface hybrid, using the control gene LTA, 107 bases long, as a model target. An array of 15 gold electrodes was used to detect the formation of hybridised duplex following interaction of non-hybridised guanine bases with MB present in solution. Upon hybridisation the number of free guanines present at the electrode surface increased from 8 to 25 due to guanine bases present in the target sequence which did not participate in hybridisation and remained free to interact directly with methylene blue. This increase in free guanines consequently concentrated MB directly at the electrode surface. We found that the MB signal recorded for 100 nM of the complementary LTA was typically 2.14 times higher than that of the non-hybridised state. Very low cross-reactivity (<7%) with a non-complementary probe was recorded. The assay was optimised with regards to methylene blue concentration, hybridisation time and regeneration. The assay was quantitative and linear in the range of 6.25-50 nM target DNA exhibiting an LOD of 17.5 nM. The assay was rapid and easy to perform, with no need for lengthy incubations with the methylene blue label or requirement for washing steps. Ongoing work addresses the impact of guanine location on the signal in order to tailor design specific signalling domains of PCR products.

Surface plasmon resonance sensor for domoic acid based on grafted imprinted polymer.

A molecularly imprinted polymer (MIP) film for domoic acid (DA) was synthesised by direct photo-grafting onto a gold chip suitable for a surface plasmon resonance (SPR) based bioanalytical instrument system, the BIAcore 3000. The gold surface was first functionalised with a self-assembled monolayer of 2-mercaptoethylamine and subsequent carbodiimide chemistry was performed for covalent attachment of the photoinitiator, 4,4'-azobis(cyanovaleric acid). This ensured that the formation of the MIP thin film, comprising 2-(diethylamino) ethyl methacrylate as functional monomer and ethylene glycol dimethacrylate as cross-linker, occurred only at the surface level. Optimisation and control over the grafting procedure were achieved using contact angle measurements and atomic force microscope (AFM) imaging. The surface grafting resulted in the formation of thin and homogeneous MIP film with thickness of 40 nm. A competitive binding assay was performed with free DA and its conjugate with horseradish peroxidase, which was used as a refractive label. The sensor was evaluated for its sensitivity, cross-reactivity, and robustness by using a BIAcore 3000. Likewise, monoclonal antibodies acting as natural receptors for the toxin were studied with the same BIAcore system. Results of a comparison between the artificial and natural receptors are reported. In contrast to monoclonal antibodies, the regeneration of MIP chip did not affect its recognition properties and continuous measurement was possible over a period of at least 2 months.