Fluorescent microspheres lateral flow assay for sensitive detection of the milk allergen casein

Figures & data

Figure 1. (a) Competitive format structure of lateral flow assay. (b) The actual picture images of negative and positive test results of CG-LFA and FMs-LFA.

Figure 2. Standard curve of CG-LFA and FMs-LFA constructed by plotting B/B₀ ratio against the concentration of casein. Error bars represent standard deviations from the means (n = 3).*p < .05 for the B/B₀ ratio of CG-LFA versus that of FMs-LFA. #p < .05 for the B/B₀ ratio of FMs-LFA versus that of CG-LFA.

Table 1. Three spiked concentrations (1000, 5000, and 10,000 ng/mL) of casein were analyzed for intra- and inter-assay recovery studies of the FMs-LFA and the CG-LFA.

Spiked casein (ng/mL)	Intra-assay^a						Inter-assay^a
	FMs-LFA			CG-LFA			FMs-LFA			CG-LFA
	Recovery (%)	SD	CV (%)	Recovery (%)	SD	CV (%)	Recovery (%)	SD	CV (%)	Recovery (%)	SD	CV (%)
1000	89.03	0.02	5.2	98.47	0.07	7.1	87.69	0.05	6.2	105.6	0.11	10.4
5000	93.47	0.07	6.9	105.3	0.11	10.4	92.73	0.16	8.3	103.3	0.13	12.6
10,000	91.2	0.23	7.8	79.24	0.06	7.6	89.82	0.29	8.5	85.72	0.09	10.5

^aData are the means of triplicates.

Figure 3. Specificity study of FMs-LFA. Concentrations of casein, lysozyme, β-lactoglobulin, BSA, and OVA were 4.0 μg/mL (A), 7.0 μg/mL (B), and 9.0 μg/mL (C), respectively. and the 0.01 M of PBS buffer was used as the negative control. Error bars represent standard deviations from the means (n = 3).*p < .05 for the S_T/S_C ratio of casein versus that of the four other proteins and PBS.

Table 2. Comparison of detection results between FMs-LFA and ELISA kit.

Sample	ELISA kit (ng/mL)			FMs-LFA (ng/mL)
	Mean^a (ng/mL)	SD	CV (%)	Mean^a (ng/mL)	SD	CV (%)
1	6035.0	211.2	3.5	6193.3	263.2	4.2
2	1114.7	97.4	8.7	1328.0	71.6	5.4
3	729.3	31.7	4.3	722.7	41.1	5.7
4	1376.1	93.7	6.8	1451.2	90.3	6.2

^aMean values of triplicate measurements.

Table 3. Detection result of casein in three kinds of milk samples.

Milk sample^a	Spiked concentration^b (ng/mL)	Detected concentration (ng/mL)	Recovery^c (%)
Ultra-heat-treated milk	0	715.2 ± 25.2
	200	867.1 ± 41.1	94.7
	500	1174.8 ± 110.3	96.7
	2000	2779.6 ± 124.3	102.4
	6000	7161.4 ± 431.2	106.6
Ultra-heat-treated skimmed milk	0	558.3 ± 21.5
	200	762.6 ± 34.3	100.5
	500	931.6 ± 56.8	88.9
	2000	2593.8 ± 153.1	101.4
	6000	6752.4 ± 389.2	103.5
Pure goat milk	0	329.3 ± 18.9
	200	520.1 ± 31.2	98.3
	500	852.6 ± 49.6	102.8
	2000	2227.8 ± 1 63.7	95.6
	6000	6624.8 ± 586.2	104.7

^aEach milk sample was diluted to 1 × 10⁴ times with 0.01M PBS buffer, then spiked different concentrations of casein and detected using the test strip.

^bData are the means of triplicates.

^cRecovery = detected concentration/(spiked concentration + background concentration in sample).