Gold nanoparticle-based enhanced lateral flow immunoassay for detection of Cronobacter sakazakii in powdered infant formula

Abstract

Cronobacter sakazakii is an opportunistic foodborne pathogen that can infect newborns through powdered infant formula (PIF). In this study, we developed a novel enhanced lateral flow immunoassay (LFA) with enhanced sensitivity for detection of C. sakazakii in PIF by the naked eye. The proposed strategy for signal enhancement of the traditional LFA used concentrated gold nanoparticles (AuNP) as the enhancer to conjugate with capture antibodies, which could increase the immobilized capture antibodies concentration at the detection zone to improve capture efficiency. Besides, the detection signal was further amplified by accumulated AuNP as the C. sakazakii labeled with AuNP probes was captured by antibodies conjugated with enhancer at the test line. We also studied the effect of different concentrations of capture antibodies and concentrated AuNP on detection performance, and found that 2.2 mg/mL of capture antibodies and 0.06 nM concentrated AuNP were the optimal combination that could avoid a false-positive signal and maximally amplify the detection signal of the enhanced LFA. Using this strategy, the detection sensitivity of the enhanced LFA was 10 cfu/mL and improved 100-fold compared with traditional LFA. The strip was highly specific to C. sakazakii, and the time for detection of C. sakazakii in PIF was shortened by 3 h. In summary, the enhanced LFA developed by the addition of concentrated AuNP as the enhancer can be used as a sensitive, rapid, visual qualitative and point-of-care test method for detecting target analytes.

Immunogold Nanoparticles for Rapid Plasmonic Detection of C. sakazakii

“…Our proof of concept has the advantage of presumably leading to a low-cost and rapid assay since low-cost spectrometers for a few hundred Euros provide sufficient sensitivity and the tests can be rapidly performed on site by lesser-trained personnel. For example, an LFA strip-based readout, as mentioned in the introduction, can be even lower cost and easier to use, but at the expense of a higher limit of detection [ 18 ]. PCR analysis requires lab equipment that are orders of magnitude more expensive plus they require more time of skilled personnel.…”

“…The LFA strip using a sandwich assay of Au nanoparticles with monoclonal antibodies specific to C. sakazakii , achieved assay times after preparation of the strips and nanoparticles significantly shorter than 1 h using a very simple readout format that can be used anywhere. However, the detection limit was probably bounded by the background signal from nonspecific binding and especially by the less efficient targeting obtained from a macroscopic surface compared to targeting in the bulk using nanoparticles; hence the limit of detection was two orders of magnitude higher than in our assay at >10 3 CFU/mL [ 18 ]. A similar detection limit of ~10 3 –10 4 CFU/mL was achieved using magnetic immunoliposomes and a fluorescence-based readout [ 8 ].…”

“…Such “immunogold” particles have long been a staining technology for biochemical identification in electron microscopy, since they in principle allow for imaging of the distribution of biomolecules on e.g., cell membranes [ 16 , 17 ]. The advantages of nanoparticle-based antibody labels, such as high optical (plasmonic extinction and surface-enhanced Raman scattering) signals, magnetic properties, increased affinity, more rapid and efficient binding than surface-based methods and high electron density contrast for electron microscopy imaging, have made them increasingly attractive to be used as biochemical sensors, nanoscale building blocks and immunohistochemical probes [ 18 , 19 , 20 , 21 ]. Several novel detection schemes have been proposed that utilize the special properties of the nanometric inorganic particles.…”

“…This approach could detect <10 4 CFU/mL within 2.5 h and the measurements had a linear range up to 10 6 CFU/mL as a function of the logarithm of concentration. In another recent report, a lateral flow immunoassay (LFA) was developed for equally sensitive detection of C. sakazakii , which took advantage of an immunogold sandwich assay to improve sensitivity [ 18 ]. Using gold nanoparticles functionalized with monoclonal antibodies, the LFA strip test could be made highly specific to C. sakazakii with a detection limit of almost 10 3 CFU/mL (two orders of magnitude more sensitive than standard LFA) and a detection time shortened by 3 h compared to other LFA.…”

Desarrollo de métodos inmunoquímicos para la determinación de sustancias tóxicas en alimentos y aguas

“…[101][102] Una forma de caracterizar la interacción antígeno-anticuerpo es su evaluación desde los puntos de vista cinético y termodinámico. Así, los parámetros de mayor interés que pueden proporcionar estudios de este tipo son las constantes de velocidad y de equilibrio asociadas al 132 Un gran número de publicaciones científicas en el área del diagnóstico de la medicina, [133][134][135] , veterinaria, 135 en el control de drogas, 136 análisis medioambiental [137][138] o en la seguridad alimentaria, [139][140][141][142][143][144][145] han sido un claro ejemplo de la importancia que han alcanzado los métodos inmunocromatográficos durante los últimos años.…”