Thesis presented October 14, 2016

Abstract:
The usual laboratory technique to quantify blood biomarkers is the enzyme-linked immunosorbent assay (ELISA) ^[10]. The method is used for different purposes, among them diagnosis and therapy monitoring. This immunological method is based on the use of two different antibodies (for the sandwich ELISA) aimed at the analyte to be quantified. The first antibody is immobilized on solid substrate, and the second is chemically coupled to an enzyme. The enzyme's product, a colored or fluorescent molecule is measured by spectrophotometry and serves as a signal amplifier to quantify the analyte. Sample preparation steps are required due to the detection method employed. Cells have to be removed by centrifugation, and the resulting plasma is then diluted. Rapid immuno-assay had been developed in the form of point-of-care tests (POC), e.g. tests to be performed at the patient bedside, or simple and low costs tests for developing countries. Among the rapid immune-assays is the lateral flow immuno-assay, a well-known application is the pregnancy test. In this technique ^[6] a strip of porous material perform a complex biological protocol (capturing, marking, washing) and fluidic actuation Immuno-agglutination of red blood cells was also considered as the basis of rapid assay for specific antigen detection. These assay use bispecific antibodies aimed towards both the red blood cell and the targeted analyte. If the analyte, that must have two separated binding sites (like for the sandwich ELISA), is present in a patient's blood, red blood cells agglutinates will form mediated by bridging from the analyte and bispecific reagent. Using this technique rapid detection (less than 15 minutes) of Hepatitis B surface antigen had been achieved from 20 µL of blood ^[2]. Another study had shown the detection of anti-HIV antibodies by the same techniques with sensitivity and specificity over 95% ^{[3, 8]}. All of these tests were final point study with a completely manual procedure and there limit of detection remain way below the ELISA The goal of the thesis is to study the feasibility of the quantification of an analyte by performing an immuno-agglutination assay mediated by a bi-specific reagent. It must be possible to perform the assay using undiluted whole blood to quantify a protein, a particle or a virus? The protocol will be as automated as possible using a simple passive microfluidic chip, the measurement of agglutination will rely on a lensless imaging device ^{[1, 4]}, consisting only in a low-cost CMOS sensor and a light source. This technological choice is particularly well adapted to perform bedside testing because of the reduce cost and bulk of the instrument compared to lens based optical instruments, the latter would also require very precise mechanical assembly. The imaging technique allows dynamic monitoring of the agglutination reaction. During the thesis it is expected to develop an automated and autonomous protocol and instruments capable of quantifying an analyte concentration in blood using red blood cell agglutination. The protocol is based on the embedding of the reagents in a passive microfluidic chip, and the instrument is based on lensless imaging and image processing algorithms in order to compute the concentration of the analyte.
^[1] Allier et al. (2010) Biomed Opt Expr 1(3): 762-770
^[2] Chen et al. (2007) Clin Vacc Immunol 14(6): 720-725
^[3] Gupta et al. (2003) J Clin Microbiol 41(7): 2814-2821
^[4] Isikman et al. (2012) Anal Cell Path 35(4): 229-247
^[5] Pingel et al. (2012) Biosens Bioelectron 31(1): 554-557
^[6] Posthuma-Trumpie et al. (2009) Anal Bioanal Chem 393(2): 569-582
^[7] Reisser-Rubrecht et al. (2008) Chem Res Tox 21(2): 349-357
^[8] Shao et al. (2008) Cell Mol Immunol 5(4): 299-306
^[9] Sollier et al. (2010) Biomed Microdev 12(3): 485-497
^[10] Voller et al. (1978) J Clin Path 31(6): 507-520

Keywords:
Microfluidic, Point-of-care, blood, agglutination, quantification, image processing