A generic sandwich-type biosensor with nanomolar detection limits

Abstract

A quantitative and highly sensitive, yet simple and rapid, biosensor system was developed for the detection of nucleic acid sequences that can also be adapted to the detection of antigens. A dipstick-type biosensor with liposome amplification, based on a sandwich assay format with optical detection, was combined with a simple coupling reaction that allows the transformation of the generic biosensor components to target specific ones by a mere incubation step. This biosensor platform system was developed and optimized, and its principle was proven using DNA oligonucleotides that provided a nucleic acid biosensor for the specific detection of RNA and DNA sequences. However, the coupling reaction principle chosen can also be used for the immobilization of antibodies or receptor molecules, and therefore for the development of immunosensors and receptor-based biosensors. The generic biosensor consists of liposomes entrapping sulforhodamine B that are coated with streptavidin on the outside, and polyethersulfone membranes with anti-fluorescein antibodies immobilized in the detection zone. In order to transform the generic biosensor into a specific DNA/RNA biosensor, two oligonucleotides that are able to hybridize to the target sequence were labeled with a biotin and a fluorescein molecule, respectively. By simultaneously incubating the liposomes, both oligonucleotides, and the target sequence in a hybridization buffer for 20-30 min at 42 degrees C, a sandwich complex was formed. The mixture was applied to the polyethersulfone membrane. The complex was captured in the detection zone and quantified using a hand-held reflectometer. The system was tested using RNA sequences from B. anthracis, C. parvum and E. coli. Quantitation of concentrations between 10 fmol and 1000 fmol (10-1000 nM) was possible without altering any biosensor assay conditions. In addition, no changes to hybridization conditions were required when using authentic nucleic acid sequence-based amplified RNA sequences, and the generic biosensor compared favorably with those previously developed specifically for the RNA sequences. Therefore, the universal biosensor described is an excellent tool, for use in laboratories or at test sites, for rapidly investigating and quantifying any nucleic acid sequence of interest, as well as potentially any antigen of interest that can be bound by two antibodies simultaneously.

Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey

“…Labels are made of coloured or fluorescent nanoparticles with sizes of 15-800 nm, allowing an unobstructed flow through the membrane. They are often made of colloidal gold [1,[17][18][19][20] or latex [21], less often selenium [22], carbon [2,23] or liposomes [24][25][26][27][28][29] are used. Latex nanoparticles are coloured [21] for optical detection.…”

Evaluation of the Antimicrobial and Antioxidant Activities of Origanum dictamnus Extracts before and after Encapsulation in Liposomes

“…In recent studies [13,14], nanoencapsulation generally improved the antimicrobial activity of compounds and maintained the stability of antimicrobials over prolonged periods of time, thereby improving the safety of fresh and processed food products. Development of release-on-demand, targeted nanoencapsulation systems has implications beyond the delivery of bioactive compounds and may lead to the development of new invivo intracellular and cell membrane biosensors with the ability to monitor growth and/or kill of pathogens and spoilage organisms in real time [4]. Based on results of studies of liposomes for pharmaceutical and medical uses, food scientists have begun to utilize liposomes for controlled delivery of functional components such as proteins and enzymes, vitamins and flavors in various food applications [31].…”

Recent advances in the development of nucleic acid diagnostics

“…These forms of assays are well characterized for immunoassays and form the basis of the well established sandwich assay format. Sandwich assays have also been described in nucleic acid diagnostics [51].…”