Novel assays for detection and characterization of soluble proteases

• Main Author: Goyal, Garima
• Other Authors: Bo Liedberg
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2021
• Subjects: Engineering::Materials; Science::Chemistry
• Online Access: https://hdl.handle.net/10356/146589
• Institution: Nanyang Technological University

Protease belongs to a class of enzymes that catalyze the hydrolysis of peptide bonds between amino acids, within peptides and proteins. It is involved in cellular homeostasis by performing functions as primary as food degradation to regulation of other proteins by their precise cleavage. Little aberrations in its expression can lead to the development of different diseases. Hence, the differential expression levels of protease offer opportunities to employ it as a disease biomarker. This thesis focusses on the development of next generation assays that enable sensitive, specific, and rapid detection of disease biomarkers. The assays of prime interest are functional assays, which are based on a mechanistic action of the target enzyme (protease) over the substrate molecule. They are preferred over affinity assays as protease activity can be regarded as an indicative of disease’s progression. Furthermore, the overarching ambition is to transfer the assays from the lab setup into a portable format for point of care (POC) testing or field use. The emphasis was on Matrix Metalloproteinase-7 (MMP-7), one of the simplest proteinases in a 24-member family of matrix metalloproteinases. It is involved in precise regulation of ECM degradation, and is known to be highly upregulated in pancreatic, colorectal and lung cancer. MMP-7 in its mature form was recombinantly expressed in E.Coli and was purified in high yields using refolding of inclusion bodies. To develop a functional protease assay, a peptide substrate that is readily cleaved by MMP-7 is required. Therefore, a previously established 42-mer helix-loop-helix peptide (JR2EC) with two cleavage sites for MMP-7 was employed, initially. Better peptides were subsequently identified by screening random peptide libraries using a high-throughput technique named Self-Assembled Monolayer for Matrix-Assisted Laser-Desorption–Ionization Mass Spectrometry (SAMDI-MS). This random array was screened for peptides immobilized on gold surface and a peptide with a 7-fold better catalytic efficiency was identified and further tested. In addition, this thesis also describes the use of gold nanoparticles (AuNPs) and gold-silver alloy nanoparticles (Au0.8Ag0.2NPs) for naked-eye detection of MMP-7. These NPs were employed in an aggregation assay format using JR2EC for colorimetric detection of MMP-7. The Au0.8Ag0.2NPs were found to be superior and yielded a visual limit of detection (LOD) in a homogeneous solution that was 5 times lower (0.5 µg/mL or 25 nM) than that for AuNPs (2.5 µg/mL or 125 nM) of similar size. The performance of the MMP-7 assay was further explored using a smartphone and RGB analysis to yield a highly competitive LOD of 0.082 µg/mL or 4nM. Furthermore, an MMP-7 driven AuNP aggregation assay on paper membrane was demonstrated and it exhibited a colorimetric response with a LOD that was ~4 times lower than the corresponding LOD observed for a similar assay designed to work in homogeneous solutions. The paper membrane assay was later translated into a syringe actuated assay format with integrated filtration membranes to assist in the removal of interfering molecules present in complex matrices. This enabled the rapid detection of MMP-7 in human plasma at a highly competitive LOD (2 µg/mL or 100 nM). In addition to the advantageous optical properties of Au0.8Ag0.2NPs, the incorporation of silver also decreased the undesirable adsorption of proteinaceous matrix interferences on the NP surface (~5 times lesser non-specific binding on Au0.8Ag0.2NPs compared to AuNPs). This was utilized to develop a nucleic acid assay where the Au0.8Ag0.2NPs to PNA ratio was tuned for human vision perception. This homogeneous assay enabled a rapid visual detection of as low as 200 pM (1.4 ng/mL) and 3 nM (21 ng/mL) of nucleic acid in buffer and plasma extract, respectively, which is a substantial improvement (~250 times) as compared to previously reported LODs. The above assays for clinically relevant detection of protease and nucleic acid in complex matrices hold the potential to be translated into simple, robust, easy-to-use, and rapid assays for early diagnosis of medical complications in a clinical setting, patient’s home or in resource limited regions. Thus, the development of these assays offers attractive and affordable healthcare solutions.