Biomarker detection for global infectiuos diseases based on magnetic particles

Resumen

Infectious diseases are becoming a major threat worldwide due to the fast dissemination and adaptation of pathogens favored by the unrestricted globalization. The primary role of diagnostics is to identify a disease. The rapid identification of a disease allows the patient to be placed on a specific antimicrobial therapy and avoid prolonged management on empiric, potentially inappropriate drug. Therefore, point-of-care (POC) devices that can reliably detect and/or monitor diseases would result in an improved care, and minimization of patient and societal cost of illness. Among them, electrochemical biosensors have the advantage of high sensitivity/specificity as well as simplicity of instrumentation, and can be easily expanded to multiplex detection platform. Furthermore, the integration of magnetic particles (MPs) in POC tests provides an even increased sensitivity and specificity due to the isolation and preconcentration of the target, whether MPs are modified with a specific recognition biomolecule. Modified-MPs can thus specifically bind the biomarkers and preconcentrate them from the complex specimen under magnetic actuation, preventing interferents before testing. Affordable emerging technologies requiring minimal training for final users, such as magnetic actuated electrochemical biosensors, are presented in this dissertation. Firstly, two simple diagnostic tests for CD4+ T lymphocytes quantification, directly in whole blood, and based on magnetic particles are presented. The assay is performed in an ELISA-like format for the optical detection or using graphite-epoxy electrodes for the electrochemical biosensing strategy. In both cases, the strategy has involved three main steps: a) immunomagnetic separation of CD4+ cells by antiCD3-MPs and labeling by using biotinylated antiCD4 antibody; b) enzymatic labeling; and c) detection based on the peroxidase activity. The dual labeling (CD3 and CD4 receptor) not only avoids interferences of other cells, but also increases the specificity of the assay. Thus, the development and evaluation of magnetic-actuated rapid HIV diagnostic platforms appropriate for their use in low resource settings for the following-up of patients under treatment is demonstrated. Secondly, an interferon-gamma release assay based on electrochemical detection for interferon-gamma transcript detection produced by isolated T lymphocytes is described. This approach also involves the integration of MPs for the isolation and preconcentration of three different targets (including whole T lymphocytes, mRNA transcripts and double-tagged DNA) in the same test. Accordingly, T lymphocytes are isolated from whole blood using antiCD3-MPs. Secondly, mRNA presenting poly(A) tail is preconcentrated on polydT-MPs from T lymphocyte. Afterward, mRNA is retrotranscripted and cDNA amplified by multiplex double-tagging PCR for the specific amplification of IFN-gamma and GAPDH. Finally, one of the tags of the primers is used for the amplicons immobilization on streptavidin-MPs as support, while the electrochemical magneto-genosensing for transcript detection is performed using the other tag. This strategy results in an alternative for IFN-gamma release assays, which can be used for identifying infectious states such as Tuberculosis. Finally, the design of a diagnostic test involving a rapid, specific and highly sensitive procedure based on isothermal amplification on MPs with electrochemical readout is presented. Isothermal amplification techniques are emerging as good candidates to replace PCR for the identification of infectious microorganism, since PCR-based method can be a critical barrier in low resource settings. An electrochemical DNA detection using padlock probes and the subsequent amplification with rolling circle and circle to circle amplification is presented in Chapter 6. This strategy has demonstrated to be a powerful combination for highly specific and sensitive nucleic acid detection that can be applied in clinical diagnosis. The electrochemical biosensors developed in this dissertation, offers considerable promise for obtaining information in a faster, simpler and cheaper manner compared to traditional methods for infectious disease diagnosis. Moreover, the strategies possess great potential in many applications, in low resource settings.