研究目的
To develop a fast and efficient biosensor for the screening of milk samples contaminated by Salmonella spp. and provide a quick and cost-effective method as an alternative to the time-consuming conventional cultivation- or PCR-based approaches.
研究成果
The developed electrochemical immunosensor combining the specificity of magneto-immunocapturing with the sensitivity of electrochemical detection enabled analysis in a limited sample volume, and was able to detect the contamination of milk samples by Salmonella spp. cells in count as low as 4 CFU/mL. Owing to its simplicity and timelessness, this biosensor may serve as a reliable tool for the rapid screening of bacterial contaminants in milk.
研究不足
The complexity of food samples, such as milk matrix which contains high concentration of nutrients and cells, can negatively influence the detection of pathogens. Moreover, it is necessary to ensure compliance with the legislation mandated by the international organization for standardization (ISO).
1：Experimental Design and Method Selection:
The study involved the magneto-immunocapturing of target bacterial cells together with specific labelling using an electrochemically active probe. The subsequently created immunocomplexes were magnetically separated and analyzed by square-wave anodic stripping voltammetry (SWASV).
2：Sample Selection and Data Sources:
Salmonella enterica subsp. enterica Serovar Typhimurium ATCC 43971 was cultured on nutrient agar No. 2 at 37 °C. For immunomagnetic separation, the culture was resuspended in a physiological solution.
3：List of Experimental Equipment and Materials:
Sodium borohydride, trisodium citrate, cadmium chloride, sodium tellurite, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), the sodium salt of N-hydroxysulfosuccinimide (sulfo-NHS), 2-(N-morpholino)ethanesulfonic acid (MES), bovine serum albumin (BSA), Tween 20, bismuth(III) nitrate pentahydrate, Polyester 2,2-Bis(hydroxyl-methyl)propionic acid (bis-MPA) dendron with 8 carboxyl and 1 amine (generation 3) (MPA dendron 8C/1A), specific antibodies: polyclonal goat anti-Salmonella spp. IgG, ProMag? 1 Series COOH Surfactant-free superparamagnetic particles (
4：745 μm). Experimental Procedures and Operational Workflow:
Magnetic immunosorbent was mixed with suspension containing Salmonella CFUs/mL in PBS-Tween buffer or UHT whole milk. The electrochemically active probe was added, followed by incubation. The resulting complexes were magnetically separated and washed. Electrochemical measurements were performed using a portable electrochemical interface PalmSens connected with screen-printed three-electrode sensors with a carbon working, a carbon auxiliary, and a silver pseudoreference electrode modified by an in-situ formed bismuth film.
5：Data Analysis Methods:
The Cd(II) ions released from the core of QDs were monitored by SWASV after the acidic dissolution of immunocomplexes in hydrochloric acid. The current response provided by Cd(II) ions is directly proportional to amount of Salmonella spp. cells present in the sample.
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