Highly labeled methylene blue-ds DNA silica nanoparticles for signal enhancement of immunoassays: application to the sensitive detection of bacteria in human platelet concentrates

Abstract

A nanoparticle-based electrochemical sandwich immunoassay was developed for bacteria detection in platelet concentrates. For the assay, magnetic beads were functionalized with antibodies to allow the specific capture of bacteria from the complex matrix, and innovative methylene blue-DNA/nanoparticle assemblies provided the electrochemical response for amplified detection. This nanoparticular system was designed as a temperature-sensitive nano-tool for electrochemical detection. First, oligonucleotide-functionalized nanoparticles were obtained by direct synthesis of the DNA strands on the nanoparticle surface using an automated oligonucleotide synthesizer. Densely packed DNA coverage was thus obtained. Then, DNA duplexes were constructed on the NP surface with a complementary strand bearing a 3 methylene blue tag. This strategy ultimately produced highly functionalized nanoparticles with electrochemical markers. These assemblies enabled amplification of the electrochemical signal, resulting in a very good sensitivity. A proof-of-concept was carried out for E. coli detection in human platelet concentrates. Bacterial contamination of this complex biological matrix is the highest residual infectious risk in blood transfusion. The development of a rapid assay that could reach 10-102 CFU mL-1 sensitivity is a great challenge. The nanoparticle-based electrochemical sandwich immunoassay carried out on a boron doped diamond electrode proved to be sensitive for E. coli detection in human platelets. Two antibody pairs were used to develop either a generic assay against certain Gram negative strains or a specific assay for E. coli. The methylene blue-DNA/nanoparticles amplify sensitivity ×1000 compared with the assay run without NPs for electrochemical detection. A limit of detection of 10 CFU mL-1 in a biological matrix was achieved for E. coli using the highly specific antibody pair.

Point-of-Need DNA Testing for Detection of Foodborne Pathogenic Bacteria

“…Electrochemical detection involves the interaction of chemical species with electrodes carrying an immobilized probe, such as DNA. Electrochemical detection on microchip platforms has become a popular technique for implementation in the field-portable devices [70,154,155,156,157,158,159,160]. Furthermore, electrochemical detection is one of the best alternatives to optical detection due to its inherent sensitivity, capability to be miniaturized without loss of performance, and high compatibility with microfabrication techniques.…”

Highly Sensitive Detection ofCampylobacter spp.in Chicken Meat using a Silica Nanoparticle Enhanced Dot Blot DNA Biosensor

“…Biotin-Si-NPs were prepared by an innovative solid-phase synthesis technology that allowed for a very high functionalization. Hydroxyl group was attached to the Si-NP surface by silanization to support the oligonucleotide linker synthesis on the NP surfaces, as previously reported (Bonnet et al 2018;Farre et al 2010). In the final step, biotin groups were grafted to the linker.…”

“…Si-NPs was functionalized by an innovative solid-phase synthesis technology which enables functionalization of nanosized particles with DNA fragments, as reported previously (Bonnet et al 2018;De Crozals et al 2012). Briefly, the nanoparticle immobilization onto controlled pore glass (CPG) allows a very high functionalization with a modified oligonucleotidic based linker.…”

“…The amount of linker grafted to nanoparticles was quantified by measuring absorbance in water (200 µL) at 260 nm and 560 nm with a microplate reader (Perkin Elmer). The nanoparticle concentration was estimated as described previously (Bonnet et al 2018). To fit with this estimation, we approximated a molar extinction coefficient to 163200 M -1 cm -1 that considers the epsilons of the different parts of the sequence corrected by their corresponding coupling yield.…”

Challenges in Electrochemical Aptasensors and Current Sensing Architectures Using Flat Gold Surfaces

“…To tackle the problem of low signals in micro/nanoelectrodes, it is necessary to build more sensitive electronics [28] or develop better signal sources. Following the latter, Bonnet and coworkers [34], developed a probe bearing three MB molecules that can be coupled into a DNA strand during standard solid-phase synthesis. This novel poly-MB probe was used to detect bacteria in a sandwich assay using gold nanoparticles and microorganisms captured on magnetic beads.…”