Los métodos de detección rápida con alta sensibilidad, selectividad y especificidad en entornos complejos son esenciales para la detección de patógenos bacterianos. En un artículo reciente publicado en la revista Analytical BiochemistryLos investigadores desarrollaron un nuevo inmunosensor de transferencia de energía por resonancia de fluorescencia (FRET) para la detección Helicobacter pylori (H. pylori) bacterias con alta sensibilidad.
Study: Carbon dots and graphene oxide based FRET immunosensor for sensitive detection of Helicobacter pylori. Image Credit: K_E_N/Shutterstock.com
Highly fluorescent and water-dispersible functionalized carbon dots (FCDs) were synthesized from an organic source, followed by the fabrication of a potential fluorescence probe (FCDs-Ab) by conjugating the FCDs with anti-H. pylori antibody. The fluorescence quenching caused due to interactions between FCDs-Ab and graphene oxide (GO) was restored due to the presence of H. pylori.
Characterization of the FRET immunosensor after each step exhibited a linear detection range of 5 x 107 cells per milliliter, and the limit of detection (LOD) was 10 cells per milliliter for H. pylori. The analytical studies on developed FRET immunosensor using spiked food samples revealed decent recovery rates with promising risk assessment capacity in food testing.
H. pylori and Fluorescent Biosensing Methods
H. pylori is a pathogenic bacterium causing gastrointestinal (GI) tract infections in the human population, sometimes leading to permanent damage to the GI tract. According to World Health Organization (WHO), H. pylori is a class I carcinogen that colonizes the stomach lining and causes liver cancer, gastritis, carcinoma, peptic and duodenal ulcer, and gastric lymphoma.
Additionally, increasing antibiotic resistance in the human population poses a challenge for optimizing a medical treatment against H. pylori infection. Hence, it is critical to develop an in vitro H. pylori detection method which allows for early infection monitoring caused via contaminated food and water sources. Although techniques like colorimetric or electrochemical sensors, polymerase chain reaction (PCR), enzyme-linked sorbent assays (ELISA), and lateral flow devices are available, these methods are laborious, time-consuming, and require skilled professionals for operation.
To this end, the FRET immunosensor that relies on energy transfer phenomena transfers optical energy from donor to acceptor. Furthermore, carbon nanodots (CDs) or nanocrystal-based biosensors enhance their sensitivity, photostability, water dispersibility, biocompatibility, and reduced toxicity. These nanomaterial-based biosensors can be effectively utilized for drug or gene delivery, metal ion or pathogenic microorganism detection, and in vivo and in vitro bioimaging.
GO is a two-dimensional (2D) carbon material with carboxylic (-COOH), hydroxyl (-OH), and carbonyl (-C=O) surface functionalization. It shows high water dispersion, easy surface modification, biocompatibility, and good physicochemical properties. Additionally, the unique thermal, mechanical, and electronic properties of GOs enable them to serve as energy acceptors in photoluminescent sensors and energy transfer reactions. Moreover, GO surface functionalization with sp2 aromatic rings induces fluorescence quenching via non-radiative dipole-dipole coupling or FRET phenomena.
CDs and GO-based FRET Immunosensor for H. pylori Detection
In the present study, the researchers developed a FRET immunosensor to quantitatively detect H. pylori wherein FCD-Ab served as fluorophore and GO served as a quencher. Characterization of this immunosensor using fluorescence spectroscopy, field emission scanning electron microscope (FESEM), and attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy confirmed the conjugation of Ab with FCD and their subsequent interaction with GO to form FCD-Ab-GO complex.
High-resolution transmission electron microscope (HRTEM) images of FCDs revealed their spherical shape and diameter of approximately 4 nanometers with uniform distribution. Dynamic light scattering (DLS) measurement of FCDs in an aqueous medium confirmed an average particle size of 3.79 nanometers. The ultraviolet-visible (UV-vis) absorption and fluorescence spectroscopy revealed absorption peaks at 332 and 230 nanometers, corroborating n- pie(π)* and π-π* energy transitions of -C=O and alkene (C=C) bonds, responsible for exhibiting fluorescence (FL).
Various dissimilar pathogens-based assays substantiated the selectivity and specificity of the FRET immunosensor. Furthermore, its performance comparison with the ELISA assay showed better cell count and lower LOD in the FRET immunosensor. Additionally, the FRET immunosensor’s practical applicability was analyzed using spiked food samples, and the results revealed its promising risk assessment capacity in real food.
To summarize, the newly developed FRET-based immunosensor could quantitatively detect the whole cell H. pylori using CDs and GO. The FCDs were synthesized efficiently from an organic source (citric acid) with a high quantum yield. Using a catalytic quantity of Tris during the synthesis of FCD enabled the retention of carboxyl groups on the FCD surface, leveraged for their conjugation with Abs.
The strong fluorescence exhibited by FCD-Ab conjugates helped confirm the successful bioconjugation. FESEM images revealed subsequent FCD-Ab attachment onto GO nanosheet, and fluorescence spectroscopy helped monitor subsequent fluorescence quenching. Furthermore, the fluorescence recovery, due to the presence of the target pathogen, was again monitored using fluorescence spectroscopy. Here, the intensity of peaks in the spectra correlated with the concentration of pathogen in the sample.
Chattopadhyay S, Choudhary M, Singh H (2022). Inmunosensor FRET basado en puntos de carbono y óxido de grafeno para la detección sensible de Helicobacter pylori. Bioquímica Analítica https://www.sciencedirect.com/science/article/pii/S0003269722002573?via%3Dihub