Immunoassay design of a fiber optic biosensor for medical and agro-food applications

Over the last decades, biosensors have emerged as innovative tools at the interface between advanced biotechnology and sensor technology. They have been used in multiple applications, ranging from agricultural industry and food safety to medical diagnosis and environmental monitoring, and they have drawn increasing commercial attention from the market. The ever increasing cost in the healthcare sector has driven the research towards biosensors that allow fast, sensitive, accurate and robust on-site detection. However, a single biosensor that fulfills all of these requirements is not yet available on the market. Surface plasmon resonance (SPR) based optical biosensors show a huge potential for multiple biosensing applications. These sensors exploit surface plasmon waves to measure local refractive index changes due to interactions between target molecules of interest and biomolecules immobilized on the sensor surface. The most commonly used SPR biosensor, commercialized by Biacore, employs a prism for SPR excitation and provides both sensitive and reliable results. However, the device is bulky and expensive, and is therefore not suited for on-site detection. An alternative approach for SPR excitation is to exploit optical fibers, which have the advantages of being affordable and small. In this thesis, a fiber-optic SPR (FO-SPR) biosensor is explored for a variety of biosensing applications. The FO-SPR biosensor is fully automated, easy to miniaturize, affordable, and reliable. Although it has been employed in antibody-based and DNA-based bioassays, it has never been fully deployed in real biological samples. The aim of this work is to investigate the capability of the FO-SPR biosensor for sensitive and rapid detection of a variety of target analytes in real matrices using immunoassays. To achieve this, two target molecules were selected based on their sizes and applications, namely progesterone (small molecule relevant to the agriculture industry) and infliximab (antibody relevant to the healthcare sector). In the agriculture industry, maximum yields are crucial to allow farmers to remain competitive. For milk production, it is critically important to detect heat in cows so that herd owners can conduct artificial insemination to attain an optimal successful pregnancy rate. Monitoring the level of progesterone is frequently employed for detection of heat. ELISA is the conventional technique for this situation, but this requires tests to be performed in a central laboratory and takes several hours to obtain results. Recently, a lateral flow immunoassay (LFIA) has been commercialized to perform on-farm tests and obtain results in 15 min. However, the sensitivity is worse than ELISA. To solve this problem a competitive immunoassay was developed using the FO-SPR biosensor for determining progesterone levels in milk. A limit of detection (LOD) of 0.5 ng/mL (1.6 nM) was achieved in standard milk. Moreover, validation of the assay was conducted with 6 bovine milk samples and benchmarked to the commercially available ELISA (cELISA) reference technology. A high correlation was obtained (Pearson correlation of 0.98 and ICC of 0.97) between both technologies. This work demonstrates that the FO-SPR biosensor is capable of detecting small molecules in the nanomolar range in a complex milk matrix within 20 minutes. In the healthcare sector, patients can benefit from techniques that enable continuous monitoring of chronic diseases and early diagnosis of diseases. Infliximab (IFX) is a therapeutic antibody used for patients with inflammatory bowel disease. This biologic agent requires therapeutic drug monitoring to allow dosage adjustment and therefore improve therapeutic outcomes. The traditional methods for detection of IFX such as ELISA, radioimmunoassay, and homogeneous mobility shift assay, require the tests to be performed in a central laboratory. Therefore, immediate dosage adjustment is not feasible. For this application, a sandwich immunoassay was developed. An LOD of 2.2 ng/ml (15 pM) was achieved in 100-fold diluted serum. The total time from sample to result was 40 min. The evaluation of the established immunoassay was tested with 5 serum samples from IFX treated inflammatory bowel disease (IBD) patients. The results show excellent agreement between the FO-SPR and the clinically validated ELISA (Pearson correlation of 0.998 and ICC of 0.983 with inter-CV being less than 10%). Since detection of IFX concentrations is usually done in patients' serum/plasma samples, this requires sample preparation from blood, which hampers the turnaround time. Thus, by establishing direct IFX detection in whole blood, sample preparation can be avoided. Furthermore, dried blood spot (DBS) sampling allows patients to collect blood sample at home and avoid visits to hospital. An advanced immunoassay was developed with a total detection time of 10 min. This short immunoassay was tested in matrices including serum, plasma, whole blood and DBS. An LOD of 0.9 ng/mL (5 pM) was achieved in 10-fold diluted whole blood and a LOD below 2 ng/mL (10 pM) was obtained in 100-fold diluted matrices listed above. The calibration curves for 100-fold diluted matrices indicated that the matrix effect is insignificant for this dilution factor. More importantly, all of the calibration curves were performed on two independent days with two batches of independently prepared FO-SPR sensor and gold nanoparticles (AuNPs) in a random manner. The statistical results indicate insignificant difference between the results generated on the two days, meaning that a new calibration is unnecessary for each batch of FO-SPR sensors and AuNPs. Finally, an immunoassay validation was carried out on 10 clinical samples from IFX-treated IBD patients and benchmarked with the clinically validated ELISA. Excellent agreement was obtained between the two systems. This thesis demonstrates the potential of the FO-SPR biosensor for a variety of applications that require specific, sensitive, rapid and robust detection with an affordable and portable device for POC diagnostics or on-site measurements.

Jaar van publicatie:2018

Toegankelijkheid:Open