Biofunctionalized nanoporous membrane/nanoparticles-based rapid and ultrasensitive sensing platform for biomolecule detection

Abstract

Biomolecule detection plays important roles in various applications including food safety detection, biomedical diagnosis, and environmental detection. Traditional biomolecule detection methods include polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) and fluorescent dye labeled detection methods. However, they are time-consuming, labor-intensive, and expensive and require sophisticated instrumentation. A simple, rapid and ultrasensitive sensing platform based on biofunctionalized nanomaterials is required to be developed. This study investigates biofunctionalized nanoporous membrane/nanoparticles based sensing platforms via electrochemical and optical detection mechanisms for nucleic acid hybridization and bacterial toxin protein detection. The whole study includes three parts. The first part was the development of nanoporous alumina membrane based electrochemical biosensor with gold nanoparticles (AuNPs) amplification and silver enhancement for deoxyribonucleic acid (DNA) hybridization detection. Nanoporous alumina membranes have the advantageous properties of high surface reaction area, which allowed huge numbers of probe DNA segments to be adsorbed on the nanopore walls by covalent bonding for target DNA hybridization detection and significantly increased detection sensitivity. Probe DNA was immobilized in nanopores by covalent bonding of chemical linkers. Target DNA hybridization in nanopores led to nanopore blockage and ion current decrease, which could be detected by electrochemical impedance spectroscopy (EIS). AuNP conjugation with silver enhancement in nanochannels could further increase the pore-blocking efficiency and consequently the detection sensitivity. The results demonstrated that AuNPs labelling and silver enhancement could significantly increase the sensitivity for DNA hybridization detection for both two complementary strands hybridization and sandwich structure assay detection. Compared with two complementary strands hybridization detection, sandwich structure assay detection was more suitable for real applications. The nanopore size effect on detection sensitivity was also explored. We found 100 nm nanopore size was optimal for DNA hybridization detection with AuNP labelling and silver enhancement. The limit of detection (LOD) was as low as single digit of pM. The second part was the development of nanoporous alumina membrane based electrochemical biosensor for monitoring botulinum neurotoxin type A (BoNT/A) light chain protease activity. In this part, green fluorescent protein (GFP) modified SNAP-25 peptides were first immobilized in nanopores by covalent bonding causing blockage of electrolyte ions passing through the nanopores. On chip cleavage of immobilized SNAP-25-GFP was analyzed via impedance spectroscopy with nanoporous substrate exposure to bacterial toxin BoNT/A light chain. The limit of detection was around 500 pM. The third part was the development of a nanoporous alumina membrane based Luminescence Resonance Energy Transfer (LRET) biosensor using upconversion nanoparticles (UCNPs) and AuNPs pairs for rapid and ultrasensitive detection of avian influenza virus H7 subtype. Both LRET processes in solution and on solid phase nanoporous alumina membrane were explored. Lanthanide-based UCNPs could absorb multiple low-energy near-infrared (NIR) photons and convert them into visible emission. They acted as donors with the advantages of biocompatibility, low toxicity, and photostability. AuNPs could act as good acceptors with the strong surface plasma absorption in the NIR-to-IR region. In this part, poly(ethylenimine) (PEI) modified BaGdF5:Yb/Er UCNPs were conjugated with the amino modified H7 capture oligonucleotide probes by glutaraldehyde linker. AuNPs were conjugated with thiol modified H7 hemagglutinin gene oligonucleotides. The UCNPs and AuNPs were brought near with a distance of 10 nm by the hybridization process between two complementary oligonucleotides. With 980 nm laser excitation, the emission energy of UCNPs was transferred to AuNPs and quenched. This solution based system could reach a low limit of detection 7 pM. Nanoporous alumina membranes based LRET biosensor was also constructed as a solid phase platform for carrying UCNPs for detection. Large surface area to volume ratio of nanoporous alumina membranes made it possible to immobilize large amount of UCNPs on nanpores by covalent bonding. An ultralow limit of detection of 300 fM was achieved for solid phase nanoporous alumina membrane based LRET platform.