Final Report Summary - ECLOBIOSENS (Electrochemiluminescent biosensor for screening of coeliac disease)
Project context
Electrogenerated chemiluminescence (also called electrochemi-luminescence or ECL) is a process whereby species generated at electrodes undergo high-energy electron-transfer reactions to form excited states that emit light. ECL has become a very powerful analytical technique and has been widely used in the areas of, for example, food and water testing, and bio-warfare agent detection, highlighting its use as a powerful tool for ultrasensitive biomolecule detection and quantification. There are commercially available clinical chemistry analysers, such as the Elecsys 1010 and 2010 by Roche Diagnostics, both of which are prohibitively expensive for developing regions. Thus, high-throughput, miniaturised biosensors based on ECL technology capable of multiplexed detection with a high-sensitivity, low detection limit as well as good selectivity and stability continue to attract the interest of the research community. Electrochemi-luminescence (ECL) of tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)32+) is a well-known detection method that provides high sensitivity with low background through the generation of an optical signal triggered by an electrochemical reaction. To trigger this signal, a sacrificial amine (usually tripropylamine, TPrA) is oxidised at the electrode surface generating a radical that reduces the Ru(II) complex to Ru(I) which is further transformed into a Ru(II) excited state which generates the luminescence. In comparison with traditional laser-induced luminescence detectors, the instrumentation for ECL detection is substantially less complicated and less expensive as the excitation laser and optical filters are eliminated. Moreover, because the ECL reaction only occurs close to the surface of an electrode, only a small sample is required, making ECL an ideal detection method for micro-devices. For example, ECL detection has been utilised in DNA microarray chips, where immobilised oligonucleotide probes are used to screen blood samples for the presence of specific target sequences.
Project objectives
The overall objective of ECLOBIOSENS was to develop a generic technological platform for point-of-care diagnostics capable of simultaneous genomic and proteomic detection with electrochemi-luminescent (ECL) transduction. As a model system, biosensor arrays for the screening of coeliac disease were developed based on the concurrent detection of nucleic acids associated with genetic predisposition (HLA typing) and protein molecular markers (auto-antibodies), facilitating an early diagnosis of coeliac disease.
This overall objective can be subdivided into the following sub-objectives:
-Evaluation of novel ECL detection schemes for the detection of coeliac disease susceptibility associated alleles (low-resolution HLA typing);
-Evaluation of ECL detection of IgA and IgG auto-antibodies associated with coeliac disease;
-Development of novel supramolecular DNA detection schemes based on ECL of ruthenium complexes and cyclodextrins;
-Development and testing of an ECL prototype instrument.
As a model system, biosensor arrays for the screening of coeliac disease were developed based on the concurrent detection of nucleic acids associated with genetic predisposition (HLA typing) and protein molecular markers (auto-antibodies), facilitating an early diagnosis of coeliac disease. Coeliac disease (CD) is an inflammatory disease of the upper small intestine and results from gluten ingestion in genetically susceptible individuals, and is the only lifelong nutrient-induced enteropathy. The small bowel abnormalities are reversed on withdrawal of gluten from the diet. CD is a familial condition with around 10-15 % of first-degree relatives being similarly affected. CD is strongly associated with other autoimmune conditions, such as diabetes mellitus (10 % of coeliacs affected) and auto-immune thyroiditis. While the gold standard for diagnosis for coeliac disease remains small intestinal biopsy, with morphology assessment, serological screening test have become increasingly more specific and sensitive and it is anticipated that these tests will soon replace the invasive and costly biopsy as a means of coeliac disease diagnosis. Serum IgA and IgG antibodies against gliadin, reticulin and endomysium (components of connective tissue) are detectable in individuals with CD. Measurements of serum IgG and IgA anti-gliadin antibodies (AGA) can be used to detect CD.
The Human Leukocyte Antigen system (HLA) is the name of the human major histocompatibility complex. This group of genes resides on chromosome 6 and encodes cell-surface antigens-presenting proteins that are involved in the immune system, autoimmunity and reproductive success. In coeliac disease, HLA typing can find application in establishing the genetic risk (predisposition). The factors linked to the coeliac disease genetic predisposition are well known and classified as DQ2 and DQ8 HLA variation. These two variations can be detected by detecting a limited number of alleles: DQA1*0501, DQA1* 0505, DQA1*0201, DQB1*0201, DQB1* 0202 and DQB1*0302.
Project tasks and outcomes
ECLOBIOSENS was divided into five main tasks:
1: Synthesis and characterisation of bioconjugates, in which ECL labels were linked to DNA probes, antibodies and nanoparticles. In addition, a novel ruthenium complex with potential ECL properties was prepared;
2: Evaluation of surface-chemistry methodologies, in which different surface-chemistry methodologies for linking DNA probes, proteins and antibodies were studied. In this task, a novel supramolecular method for the attachment of biorecognition elements based on cyclodextrin-modified surfaces is reported. In parallel, ECL instrumentation and electrode arrays were developed and tested;
3: Evaluation of novel ECL detection schemes for the detection of coeliac disease susceptibility alleles, in which detection methods based on ECL quenching were developed. The optimal quenching distance and other operational parameters of the biosensors were optimised;
4: Development of novel supramolecular DNA detection schemes based on ECL of ruthenium complexes and cyclodextrins, in which a supramolecular genosensor for detection of celiac disease susceptibility alleles was evaluated;
5: Evaluation of ECL detection of auto-antibodies associated with celiac disease, in which biosensors for amperometric and ECL detection of celiac disease autoantibodies were developed. In addition, a proof of concept exploiting a nanoparticle-based strategy for ECL signal enhancement was demonstrated.
Thus, the impact of ECLOBIOSENS is based on the combination of both detection strategies which can be used for general population screening, or just the HLA typing could be used for neonatal screening, or alternatively, just the auto-antibody detection could be used for monitoring compliance with the identified diet, or recurrence of the disease due to non-compliance.
The rapid development of ECL in both fundamentals and applications over the past several years has clearly demonstrated that it is a powerful tool for ultrasensitive biomolecule detection and quantification. This fact, combined with the recent advances in the understanding of celiac disease at both molecular and physiological levels, is expected to have a strong impact in the solution of a public health problem. ECLOBIOSENS also offers the possibility to apply the novel detection methods developed in solving several practical issues related to the well-being of the population and increased life expectancy.
Contact details
Dr Ciara OSullivan (coordinator) and Dr Mayreli Ortiz, Nanobiotechnology & Bioanalysis Group, Department of Chemical Engineering, Universitat Rovira i Virgili, Avinguda Paisos Catalans 26, 43007 Tarragona, Spain; E-mail: ciara.osullivan@urv.cat, mayreli.ortiz@urv.cat for more information.
Electrogenerated chemiluminescence (also called electrochemi-luminescence or ECL) is a process whereby species generated at electrodes undergo high-energy electron-transfer reactions to form excited states that emit light. ECL has become a very powerful analytical technique and has been widely used in the areas of, for example, food and water testing, and bio-warfare agent detection, highlighting its use as a powerful tool for ultrasensitive biomolecule detection and quantification. There are commercially available clinical chemistry analysers, such as the Elecsys 1010 and 2010 by Roche Diagnostics, both of which are prohibitively expensive for developing regions. Thus, high-throughput, miniaturised biosensors based on ECL technology capable of multiplexed detection with a high-sensitivity, low detection limit as well as good selectivity and stability continue to attract the interest of the research community. Electrochemi-luminescence (ECL) of tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)32+) is a well-known detection method that provides high sensitivity with low background through the generation of an optical signal triggered by an electrochemical reaction. To trigger this signal, a sacrificial amine (usually tripropylamine, TPrA) is oxidised at the electrode surface generating a radical that reduces the Ru(II) complex to Ru(I) which is further transformed into a Ru(II) excited state which generates the luminescence. In comparison with traditional laser-induced luminescence detectors, the instrumentation for ECL detection is substantially less complicated and less expensive as the excitation laser and optical filters are eliminated. Moreover, because the ECL reaction only occurs close to the surface of an electrode, only a small sample is required, making ECL an ideal detection method for micro-devices. For example, ECL detection has been utilised in DNA microarray chips, where immobilised oligonucleotide probes are used to screen blood samples for the presence of specific target sequences.
Project objectives
The overall objective of ECLOBIOSENS was to develop a generic technological platform for point-of-care diagnostics capable of simultaneous genomic and proteomic detection with electrochemi-luminescent (ECL) transduction. As a model system, biosensor arrays for the screening of coeliac disease were developed based on the concurrent detection of nucleic acids associated with genetic predisposition (HLA typing) and protein molecular markers (auto-antibodies), facilitating an early diagnosis of coeliac disease.
This overall objective can be subdivided into the following sub-objectives:
-Evaluation of novel ECL detection schemes for the detection of coeliac disease susceptibility associated alleles (low-resolution HLA typing);
-Evaluation of ECL detection of IgA and IgG auto-antibodies associated with coeliac disease;
-Development of novel supramolecular DNA detection schemes based on ECL of ruthenium complexes and cyclodextrins;
-Development and testing of an ECL prototype instrument.
As a model system, biosensor arrays for the screening of coeliac disease were developed based on the concurrent detection of nucleic acids associated with genetic predisposition (HLA typing) and protein molecular markers (auto-antibodies), facilitating an early diagnosis of coeliac disease. Coeliac disease (CD) is an inflammatory disease of the upper small intestine and results from gluten ingestion in genetically susceptible individuals, and is the only lifelong nutrient-induced enteropathy. The small bowel abnormalities are reversed on withdrawal of gluten from the diet. CD is a familial condition with around 10-15 % of first-degree relatives being similarly affected. CD is strongly associated with other autoimmune conditions, such as diabetes mellitus (10 % of coeliacs affected) and auto-immune thyroiditis. While the gold standard for diagnosis for coeliac disease remains small intestinal biopsy, with morphology assessment, serological screening test have become increasingly more specific and sensitive and it is anticipated that these tests will soon replace the invasive and costly biopsy as a means of coeliac disease diagnosis. Serum IgA and IgG antibodies against gliadin, reticulin and endomysium (components of connective tissue) are detectable in individuals with CD. Measurements of serum IgG and IgA anti-gliadin antibodies (AGA) can be used to detect CD.
The Human Leukocyte Antigen system (HLA) is the name of the human major histocompatibility complex. This group of genes resides on chromosome 6 and encodes cell-surface antigens-presenting proteins that are involved in the immune system, autoimmunity and reproductive success. In coeliac disease, HLA typing can find application in establishing the genetic risk (predisposition). The factors linked to the coeliac disease genetic predisposition are well known and classified as DQ2 and DQ8 HLA variation. These two variations can be detected by detecting a limited number of alleles: DQA1*0501, DQA1* 0505, DQA1*0201, DQB1*0201, DQB1* 0202 and DQB1*0302.
Project tasks and outcomes
ECLOBIOSENS was divided into five main tasks:
1: Synthesis and characterisation of bioconjugates, in which ECL labels were linked to DNA probes, antibodies and nanoparticles. In addition, a novel ruthenium complex with potential ECL properties was prepared;
2: Evaluation of surface-chemistry methodologies, in which different surface-chemistry methodologies for linking DNA probes, proteins and antibodies were studied. In this task, a novel supramolecular method for the attachment of biorecognition elements based on cyclodextrin-modified surfaces is reported. In parallel, ECL instrumentation and electrode arrays were developed and tested;
3: Evaluation of novel ECL detection schemes for the detection of coeliac disease susceptibility alleles, in which detection methods based on ECL quenching were developed. The optimal quenching distance and other operational parameters of the biosensors were optimised;
4: Development of novel supramolecular DNA detection schemes based on ECL of ruthenium complexes and cyclodextrins, in which a supramolecular genosensor for detection of celiac disease susceptibility alleles was evaluated;
5: Evaluation of ECL detection of auto-antibodies associated with celiac disease, in which biosensors for amperometric and ECL detection of celiac disease autoantibodies were developed. In addition, a proof of concept exploiting a nanoparticle-based strategy for ECL signal enhancement was demonstrated.
Thus, the impact of ECLOBIOSENS is based on the combination of both detection strategies which can be used for general population screening, or just the HLA typing could be used for neonatal screening, or alternatively, just the auto-antibody detection could be used for monitoring compliance with the identified diet, or recurrence of the disease due to non-compliance.
The rapid development of ECL in both fundamentals and applications over the past several years has clearly demonstrated that it is a powerful tool for ultrasensitive biomolecule detection and quantification. This fact, combined with the recent advances in the understanding of celiac disease at both molecular and physiological levels, is expected to have a strong impact in the solution of a public health problem. ECLOBIOSENS also offers the possibility to apply the novel detection methods developed in solving several practical issues related to the well-being of the population and increased life expectancy.
Contact details
Dr Ciara OSullivan (coordinator) and Dr Mayreli Ortiz, Nanobiotechnology & Bioanalysis Group, Department of Chemical Engineering, Universitat Rovira i Virgili, Avinguda Paisos Catalans 26, 43007 Tarragona, Spain; E-mail: ciara.osullivan@urv.cat, mayreli.ortiz@urv.cat for more information.