Study of aptamer selection methodologies for developing piezoelectric quartz crystal biosensors to detect albumin in urine, malaria and SARS protein biomarkers in serum

Abstract

Due to the high affinity and selectivity of aptamer selected from DNA library towards target proteins, the use of specific protein biomarkers to assist early diagnosis, and the recent rapid development of piezoelectric quartz crystal (PQC) biosensor technology, the research reported is focused on developing PQC biosensor using aptamers selected from a DNA library as selective coating for the detection of albumin in urine, two protein biomarkers for malaria in sera, and the determination of trace amount of SCV helicase protein from SAR in sera. Three new aptamer-PQC biosensors are developed with research conducted on aptamer selection, optimization of coating methods for aptamers on PQC, and coupling paramagnetic nanoparticle technology with PQC biosensor in flow injection analysis (FIA) to enrich analyte and eliminate sample matrix interference.

A new approach for aptamer selection is successfully attempted in the development of the first aptamer-PQC biosensor with an aim to offer an early detection of microalbuminuria enabled by an increase in detection sensitivity with aptamer selected by its actual binding with albumin-PQC biosensor. The aptamer-PQC sensor developed has shown to give a working range between 0.1 to 10 ug/mL, a detection limit (S/N = 3, n=3) of 0.048 μg/ml, repeatability of RSD = 6.8% (n=3), a response time of 1 minute and a throughput of 60 samples/hour. The developed piezoelectric aptamer sensor is shown to have sufficient sensitivity to detect microgram quantities of albumin in urine.

Aptamer-PQC biosensors are developed for the detection of two protein biomarkers, PfLDH for general diagnosis and PfHRP-2 for a specific diagnosis of Plasmodium falciparum malaria parasitemia, in sera. The results show satisfactory working range from 10-100 ng/mL for both PfLDH and PfHRP-2 protein biomarker, low detection limit of 1.8 and 4.7 ng/mL and satisfactory repeatability (%RSD, n = 3) of 7.4% and 9.2% for PfHRP-2 and PfLDH respectively. It is first report for aptamer based PQC biosensor to detect malarial PfHRP-2 and PfLDH at ppb range to meet the requirement for their diagnosis.

To meet the highly demanding challenge for detecting protein biomarkers at pg/mL level in high-protein sera sample, the coupling of paramagnetic nanoparticle technology with aptamer-PQC biosensor was attempted for developing aptamer-PQC biosensor for selective detection of SCV helicase protein produced from SARS CoV replication. The coupling of aptamer coated paramagnetic nanoparticles for sample pretreatment to aptamer-PQC biosensor has shown to detect helicase protein in one-minute assay with a detection limit of 350 pg/mL. The aptamer-coated crystal exhibits a frequency shift linearly proportional to the concentration of SARS helicase from 1 to 1000 ng/mL with a correlation coefficient of 0.9975 and a repeatability of 6.8% (%RSD, n=3). After the enrichment procedure, recoveries of 102% and 119% were achieved using samples spiked with SARS helicase at concentrations of 10 ng/mL and 1.0 ng/mL respectively. It is the first report to detect for SCV helicase protein using PQC biosensor at pg/mL level after magnetic bead enrichment.