A rapid abiotic/biotic hybrid sandwich detection for trace pork adulteration in halal meat extract

Experimental section

The abiotic/biotic antibody hybrid sandwich detection was performed using the following procedure: (1) PSA-MIP-NGs were

immobilized on a sensor chip, and the blocking process was

performed using 0.5% w/v BSA; (2) the cocktail solution was

prepared by mixing equal volumes of PBS containing 0.1 μg

mL−1 of Anti-PSA with 100 μg mL−1 of F-Fc-MIP-NGs; (3) PSA

was then added to the reaction mixture, followed by incubation

for 30 min; (4) the premixed cocktail solution was dropped

onto the PSA-MIP-NGs-immobilized sensor chip and incubated

for 30 min; (5) after washing with pure water (3 × 500 μL) and

PBS (3 × 500 μL), the sensor chip was inserted into a designed

flat-type pipette tip, the fluorescence intensity was measured

using a custom-made liquid-handling robot equipped with a

fluorescence microscope. The relative fluorescence intensity

was calculated using of the following equation: (F − F0)/F0,

where F0 and F are the fluorescence intensities before and

after incubation with PSA, respectively.

Meat extract samples were prepared. Chopped raw meat

(1 g) was mixed with 5 mL PBS, and then homogenized using

a benchtop homogenizer (Polytron PT 1600 E, Kinematica AG,

Luzern, Switzerland) for 2 min (10 000 rpm), followed by centrifugation for 30 min at 4 °C (3 × 16 000g). The clear supernatant was collected and filtered thrice through a 0.2 µm

polytetrafluoroethylene (PTFE) filter (DISMIC-13HP, Toyo

Roshi Kaisha Ltd, Tokyo, Japan). The filtered meat extract

samples were then used to measure total protein concentration

using a NanoDrop One UV/Vis Spectrophotometer at 280 nm.

The meat extract samples were then stored at −20 °C until use.

Author contributions

H. S. and T. T. conceived the project. C. C., H. S., E. T., and

Y. K. performed the experiments. All authors contributed to

the analysis and the interpretation of the results. The manuscript was written by C. C. and revised by H. S. and T. T. All

authors have approved the final version of the manuscript.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by JSPS KAKENHI (JP 18H05398 and

JP 23H01775) and the Research Program of “Dynamic Alliance

This journal is © The Royal Society of Chemistry 2023

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