Resumen de Study of surface chemistry strategies to enhance the electrochemical detection of proteins and DNA markers
- Justification and needs for the research Biosensors have shown many practical applications to several research fields, from medical diagnostics to environmental analysis, and have great potential for commercialization. However, despite of the great expectations, there are just few examples of commercial biosensors. This slow penetration into the market could be attributed to the elevated development and production costs and some important technological hurdles, such as sensitivity, reproducibility, matrix effects in real samples, stability and quality assurance. Taking this into account, the aim of this doctoral thesis is the development of cost-effective electrochemical detection platforms for the quantification of protein and DNA biomarkers for human diseases.
Biosensors still face a challenge in achieving a simple, robust, and inexpensive surface functionalization method compatible with mass-manufacturing techniques. Generally, functionalisation processes require the chemical modification of either the sensor surface or the capture protein or both, and this usually involves several steps that in some cases are time consuming, costly, and difficult to implement in large-scale production process. In this work, we developed an easy strategy to reduce the manufacturing cost by simplifying the surface immobilisation method of the receptor proteins to a single step, which can be applied to electrochemical, optical, and gravimetric immunosensors.
Several potential advantages, such as miniaturisation, integration, multiplexing analysis, as well as the use of low cost disposable chips, should be exploited for the biosensors to impact on the market and migrate from sophisticated laboratories to the point-of-care. Working on that direction, we also report on a procedure for the multiplex amplification and detection of several genetic markers for breast cancer using a low-density electrode microarray manufactured on standard low-cost printed circuit board (PCB) substrates.
- Methodology Various gold cleaning methods (chemical, physical and electrochemical) were developed and characterised by means of electrochemical techniques using potentiostats. Protocols assayed were based on the use of Piranha's solution, aqua regia, sodium hydroxide, hydrogen peroxide and mixtures of these as well as UV/ozone, argon plasma and cyclic voltammetry in sulphuric acid. Different surface chemistries for proteins and DNA were developed for gold electrodes. The chemistries were designed in order to maximise the specific interactions but minimise the non-specific ones to meet the required sensor sensitivity, specificity, reproducibility and multiplexing ability. Concerning the immunosensor design, the surface chemistry developed was based on the direct immobilisation on gold surfaces of disulphides-containing proteins. Disulphide groups acted as anchor molecules able to chemisorb spontaneously onto clean gold surfaces and were chemically introduced to the protein structure via the primary amines, carboxylic acids and carbohydrates present in its structure. Two proteins were used in this study, neuron specific enolase (NSE) and anti-tissue transglutamise (tTG) antibodies, an ischemic stroke and a celiac disease marker respectively. In parallel to that, immobilisation of proteins through cross-linking to self assembled monolayers (SAM)-modified gold surfaces was also assessed. The SAMs consisted in the use of alkanethiol molecules with a reactive group to couple the proteins or DNA. Regarding the genosensor development, surface chemistries for DNA immobilization based on the direct immobilization of thiolated DNA and immobilization via SAM were developed. These chemistries were applied to electrochemical sensors for the multiplexed detection of 7 genetic markers for breast cancer in tumor cells. Prior to electrochemical detection, genetic markers were amplified by means of the multiplex ligation-dependent probe amplification (MLPA) reaction, which allows for multiplex amplification of multiple targets with a single primer pair.
For each surface chemistry produced, a detection assay was developed and the assay conditions optimized. The chemistries were evaluated by optical methods such as enzyme-linked immunosorbent assay (ELISA), enzyme-linked oligonucleotide assay (ELONA) and surface plasmon ressonance (SPR), and electrochemical techniques (voltammetry, amperometry and impedance). Concerning the assay conditions, various parameters were evaluated such as receptor/backfiller ratio, target interaction time and temperature, interaction buffer, target concentration, reporter probe concentration, type of substrate and cross-reactivity of analytes. The electrochemical detection was generally based on the use a secondary reporter probe bearing a horseradish peroxidise (HRP) enzyme, which interacts with the target analyte previously captured at the electrode surface. Upon injection of the enzyme substrate, hydrogen peroxide, as well as the redox mediator 3,3',5,5'-Tetramethylbenzidine (TMB) onto the sensor surface, HRP oxidizes the TMB which was detected at the electrode surface by applying a reducing potential.
An automated procedure for the functionalisation of the electrochemical multielectrode sensors by pin-spotting using a microarrayer was developed. To stably locate the electrode array in the microarrayer printer for functionalisation, a polymethyl methacrylate (PMMA) mould was designed and fabricated.
An electrochemical sensor array was designed in-hose using technical drawing software and manufactured by an external company. The arrays were fabricated using PCB (Printed Circuit Board) technology and consist of 2-layers PCB of 1.60 mm thick with a surface finish of 3 micrometers soft Au thickness on approximately 4 micrometers Ni. Insulation layers were printed on both sides and patterned to define the final working electrode size of 300 micrometers. The electrode arrays were characterized by means of electrochemical and microscopic techniques such as scanning electron microscope (SEM) and atomic force microscope (AFM). Bare electrode exhibited well defined oxidation and reduction peak in the presence of a ferricyanide solution and voltammograms in sulphuric acid presented a well defined gold oxide region and a sharp reduction peak as can be expected from a pure gold layer. Chemical composition analysis of the working electrode surface carried out with the SEM revealed a predominant presence of gold (89 - 95 %). These results indicate the suitability of the chips for the covalent coupling of thiol- and disulfides-containing molecules.
- Conclusions It was developed a one-step method for the covalent self-assembly of antibodies on gold surfaces based on the chemical introduction of disulphides groups into the antibody structure. The surface chemistry based on the site-directed modification via the carbohydrate chains exhibited the best biosensor performance for the detection of the stroke marker NSE probably due to a better orientation of the antibody at the surface, since the sugar moieties in IgGs are specifically located on the Fc region. The same methodology was also applied for the direct immobilisation of the tTG antigienic protein in gold surfaces, which is used for the detection of the celiac disease related anti-tTG antibody. In this case, the introduction of the disulphide groups through the amine moieties exhibited the best immunosensor performance. This immunosensor was also assessed successfully with real patient samples exhibiting very low background levels, which demonstrates the suitability of the developed surface chemistry for real sample analysis. Overall, the introduction of disulphides in proteins used as bioreceptor in immunosensors provides a simple and attractive approach for a one-step covalent immobilisation, omitting the need for surface pre-treatment.
Additionally, it was developed a novel method for the multiplex barcode-MLPA-based amplification and detection of seven breast cancer related mRNA markers with single tumour cell sensitivity. The DNA amplification was performed using the barcode-MLPA approach, which enables the simultaneous amplification of multiple genes and their subsequent electrochemical detection via hybridisation of the barcode sequences to complementary surface immobilised probes. The use of barcodes enables the development of generic detection platforms, since the same barcode sequences can be used for the detection of other biomarker sets using the same surface probes. For the multiplex electrochemical detection, a low-cost electrode array was fabricated using PCB technology, which exhibited excellent conditions for biomolecules immobilisation, signal transduction and reproducibility. The developed system provides an elegant strategy for the multiplex genetic profiling of tumour cells with great possibilities for miniaturisation and integration into a stand-alone module.
