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How can we detect bacteria in Powdered Milk?

Salmonella in Poedermelk

Biosensor for Bacteria Detection from Powdered Milk
Contamination of food by pathogenic bacteria is a serious threat to human health and thus biosensors for fast and accurate food quality control are extensively studied.
Multi-Parametric Surface Plasmon Resonance (MP-SPR) based biosensor was developed to detect Salmonella Typhimurium in dairy products. Direct label-free detection of bacteria by using a capture antibody was further improved utilizing bio-catalyzed precipitation. For control samples the limit of detection (LOD) was 102 CFU/mL and for real samples (powdered milk) LOD was 103 CFU/mL, demonstrating a high sensitivity of the biosensor./mL and for real samples (powdered milk) LOD was 103 CFU/mL, demonstrating a high sensitivity of the biosensor.

Introduction
Currently used methods for Salmonella detection include culturing, enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) (Farka et al. 2016). These methods are generally timeconsuming, they require complex sample pre-treatment and trained personnel, thus more robust and easy-to-use methods are being actively developed.
Surface Plasmon Resonance (SPR) is a well-established method utilized to measure binding affinity and kinetics. Innovative Multi-Parametric Surface Plasmon Resonance (MP-SPR) instruments can perform measurements
in an exceptionally wide angular range (40-78 degrees) and at more thanone wavelength, thus allowing a wide range of applications from small molecule interactions to real-time detection of bacteria, cells and viruses.
MP-SPR is unparalleled method for biosensor development, whether your sensor is based on MP-SPR detection or whether you are developing new portable (point-of-care) biosensors. Advantages of MP-SPR are especially
high sensitivity and label-free detection. It also allows development of a sensor directly on your material-of-choice: metal electrodes for electrochemistry, plastics for well-plate assays, cellulose for printed biosensors, glass for traditional chemistry, nanoparticles for SERS, etc. avoiding assay transfer step. Sensor slides can be easily modified in situ
and ex situ providing a wide range of possibilities for functionalization (CVD, ALD, spin coating, dip coating, etc.). Additionally, MP-SPR allows measurements of real samples (milk, 100% serum, urine, sea-water, etc.),
unlike many traditional SPR instruments. After MP-SPR measurement, the sensor surface can be further characterized with other methods such as AFM. This is enabled thanks to the oil-free operation of MP-SPR, using a prism coated with an optical elastomer.

Salmonella in Poedermelk Fig2

Materials and methods
In this study, the measurements were performed with BioNavis MP-SPR Navi™ 210 VASA instrument at 20 μL/min flow-rate. Carboxymethyl dextran (CMD 3D) sensor slides were cleaned with a solution of 2 M NaCl and 10 mM NaOH
for 5 min, followed by the activation of carboxylic groups using a mixture of EDC (200 mM) and NHS (50 mM) for 7 min. The capture antibody (and BSA for the reference channel) was introduced (10 μg/mL in 50mM acetate buffer,
pH 4.5) before blocking the remaining reactive groups by ethanolamine (1 M, pH 8.5, 5 min). For additional blocking, BSA (2 mg/mL in HBS-P) and 1% powdered milk in HBS-P were injected.
The powdered milk (Laktino) was diluted in the HBS-P buffer to the concentration of 1%. Varying concentrations of Salmonella enterica (subsp.
enterica serovar Typhimurium) cells were added to the sample after culturing and heat treatment (80°C/30min). Concentrations of treated bacteria are expressed as CFU/mL corresponding to viable cells before the treatment.
The bacteria were injected for 10 min, followed by 10 min injection of the horseradish peroxidase antibody conjugate (HRP-Ab2) and 10 min of precipitation substrate solution (HRP) (Figure 1). The bio-catalyzed reaction
converted 4-chloro-1-naphthol to insoluble benzo-4-chlorocyclohexadienone.
The limit of detection (LOD) was evaluated based on the signal-tonoise ratio, where the measurable minimum signal level has to be three times higher than the noise level.

Salmonella in Poedermelk Fig3

Results and discussion
The biosensor was first developed on a Biacore 3000 SPR instrument where LOD of 104 CFU/mL was achieved. To further improve the biosensor, a bio-catalyzed precipitation reaction was selected for sensitivity enhancement. However, horseradish peroxidase requires the use of ethanol. The Biacore instrument is not compatible with alcohols, unlike BioNavis MP-SPR instruments which provide excellent resistance to organic solvents. Thus, MP-SPR Navi™ 210A VASA was selected for further research.
Using the MP-SPR instrument, a biosensor was successfully developed and the bio-catalyzed precipitation enhanced the limit of Salmonella detection (LOD) from 104 to 102 CFU/mL (Figure 2). The binding level was high in the channel containing the capture antibody and the bacteria, whereas only a minor amount of precipitate was formed in the reference channel (BSA treated). Salmonella binds multiple HRP-Ab2 conjugates which improves sensitivity of the biosensor exponentially with the increasing concentration of microbes. The HRP-Ab2 conjugate is specific to Salmonella, providing also improved selectivity compared to direct binding assay. The cross-reactivity of the developed biosensor was tested with Gram-negative bacteria E. coli K-12 which showed negligible binding. Optical microscopy and AFM images of an MP-SPR sensor slide were used as reference, and both confirmed bacteria attachment and precipitate formation on the biosensor surface (Figure 3).
The total analysis time by MP-SPR was 60 minutes which is significantly shorter than other methods used for detection of bacteria, such as cultivation (~days), ELISA (~10 h) and PCR (~hours).
Binding of bacteria is interesting not only for biosensor development but also for material sciences. MP-SPR has been utilized to characterize functional "self-defence" anti-microbial implant coatings (Cado et al., 2013). Coating releases anti-microbial peptides by stimulation with pathogens. Here, MP-SPR measures the build-up of a multilayer and quantifies adsorbed mass.
See also cancer cells (MCF7) detection from blood using a target peptide in MP-SPR instrument, Application Note #154.
Conclusions
MP-SPR proved itself as a highly sensitive and selective biosensor to detect bacteria from food samples. MP-SPR is invaluable for assay development in food and feed safety, environmental safety, clinical diagnostics, border and process control, for example.
The key benefits to use MP-SPR in biosensor development are:
Compatibility with organic solvents
Easy modification of sensor surfaces
Capability to work with crude samples
Original Publication:
Farka et al., Anal. Chem., 2016, 88 (23), pp 11830–11836
Reference:
Cado et al., Adv. Funct. Mater. 2013, 23 (38), pp 4801–4809

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