A sensitive chemiluminescent enzyme immunoassay for carbofuran residue in vegetable, fruit and environmental samples

Figures & data

Table 1. Comparison of coating effect with different coating buffers.

Buffers	pH	Ionic strength (mol/L)	Regression equation (y)	IC50 (ng/ml)	R^2	CImax
Tris–HCl	7.2	0.01	15.248 Ln(x) + 20.639	4.6	0.9433	164,984
		0.05	17.511 Ln(x) + 24.334	4.3	0.9782	187,448
		0.1	16.556 Ln(x) + 26.26	4.2	0.9753	208,396
	7.8	0.01	20.175 Ln(x) + 14.325	5.9	0.9213	110,292
		0.05	16.037 Ln(x) + 15.713	5.5	0.962	118,449
		0.1	16.872 Ln(x) + 15.146	7.9	0.9547	109,783
	8.5	0.01	18.917 Ln(x) + 17.404	5.6	0.9238	79,274
		0.05	20.23 Ln(x) + 13.291	6.1	0.961	86,740
		0.1	18.349 Ln(x) + 15.118	6.7	0.9725	72,354
PBS	6.8	0.01	15.56 Ln(x) + 10.542	12.6	0.9638	157,057
		0.05	15.783 Ln(x) + 14.427	9.5	0.9445	143,867
	7.4	0.01	17.358 Ln(x) + 22.696	4.8	0.9673	174,783
		0.05	16.866 Ln(x) + 20.453	5.8	0.99	183,934
	8.0	0.01	14.974 Ln(x) + 14.492	10.7	0.9617	129,637
		0.05	15.481 Ln(x) + 16.298	8.8	0.9562	104,822
CBS	9.6	0.05	16.317 Ln(x) + 18.346	6.9	0.9624	92,284
Distilled water	7.0	0	14.95 Ln(x) + 13.35	11.6	0.8484	298,547

Note: In the experiment, 0.05 M Tris–HCl buffer with pH 7.2 was selected as reacting buffer and 2% skimmed milk was chosen as blocking substance. Ab (6 mg/L) and BFNP-HRP (0.003 mg/L) were ascertained as working concentration. CI_max: maximal chemiluminescent intensity.

Table 2. Comparison of effect with different reacting buffers.

Buffers	pH	Ionic strength (mol/L)	Regression equation (y)	IC50 (ng/ml)	R^2	CImax
Tris–HCl	7.2	0.01	14.913 Ln(x) + 17.746	8.7	0.9233	164,984
		0.05	17.015 Ln(x) + 24.574	4.5	0.9756	218,493
		0.1	18.028 Ln(x) + 17.291	6.2	0.9721	196,832
	7.8	0.01	13.489 Ln(x) + 18.175	10.0	0.9147	139,045
		0.05	15.936 Ln(x) + 16.498	8.2	0.9639	158,633
		0.1	15.42 Ln(x) + 15.33	9.5	0.952	148,427
	8.5	0.01	13.239 Ln(x) + 18.174	11.4	0.9146	114,583
		0.05	14.734 Ln(x) + 13.697	11.7	0.9589	101,439
		0.1	13.568 Ln(x) + 14.145	14.0	0.9618	84,638
PBS	6.8	0.01	14.291 Ln(x) + 19.153	8.6	0.9713	119,382
		0.05	15.164 Ln(x) + 17.538	8.5	0.964	127,493
	7.4	0.01	17.217 Ln(x) + 23.185	4.7	0.9687	185,774
		0.05	16.837 Ln(x) + 20.324	5.8	0.9734	179,463
	8.0	0.01	15.036 Ln(x) + 17.168	8.9	0.9612	135,497
		0.05	14.73 Ln(x) + 15.165	10.6	0.9736	124,960

Note: In the experiment, 0.1 M Tris–HCl buffer with pH 7.2 was chosen as coating buffer, 2% skimmed milk was selected as blocking substance. Ab (10 mg/L) and BFNP-HRP (0.005 mg/L) were ascertained as working concentration.

Table 3. Comparison of effect with different blocking substances.

Blocking substance	Regression equation (y)	IC50 (ng/ml)	R^2	CImax
Tris–HCl	18.664 Ln(x) + 24.388	3.9	0.9278	245,467
2% Skimmed milk	17.369 Ln(x) + 23.429	4.6	0.9669	187,903
0.1% BSA	13.358 Ln(x) + 18.457	10.6	0.9664	213,690
10% FCS	14.273 Ln(x) + 18.6611	9.0	0.9543	128,573
1% Tryptone	16.249 Ln(x) + 26.513	4.2	0.9886	214,632
0.1% OVA	10.351 Ln(x) + 30.028	6.9	0.8474	170,449
1% Gelatin	15.062 Ln(x) + 25.0899	5.2	0.9545	86,389
2% Yeast extractor	14.187 Ln(x) + 21.5738	7.4	0.9632	128,752

Note: Ab (10 mg/L) and BFNP-HRP (0.005 mg/L) were ascertained as working concentration.

Figure 1. Typical standard curves for carbofuran by CLEIA under optimised conditions. Data represent the means of 10 determinations.

Table 4. Reproducibility and recovery of carbofuran from spiked samples.

Samples	Dilutions	Mean background value±SD (ng/mg, n=32)	Spiked level (ng/mg)	Mean±SD^a (ng/mg)	Mean recovery (%, n=32)	CV (%)
Cabbage	4	14±3	20.0	38±8	119.1	21.31
			100	140±26	125.63	18.33
Lettuce	2	8±2	10.0	22±5	139.1	23.13
			50.0	49±8	82.48	15.43
Carrot	1	6±2	10.0	14±3	75.7	24.28
			50.0	39±6	64.56	16.98
Pear	2	6±1	10.0	20±5	147.5	23.23
			50.0	72±14	132.3	19.84
Bamboo shoot	2	6±1	10.0	16±3	95.47	19.28
			50.0	49±9	86.66	17.41
Green soy bean	1	4±1	10.0	12±3	82.25	27.36
			50.0	52±11	95.38	20.94
Water	1	1±0.2	2.00	4±1	175.2	23.72
			10.0	13±3	124.1	22.19
Soil	1	1±0.2	2.00	4±1	148.8	26.65
			10.0	14±3	131.8	19.25

^a The mean determination value included the background value.

Figure 2. Correlation between carbofuran concentrations measured by CLEIA and by LC-MS/MS in (a) water and (b) carrot samples.

Table 5. Carbofuran residues in river water and carrot by LC-MS/MS and CLEIA.

Water			Carrot
Spiked concentration (µg/L)	LC-MS/MS (µg/L)	CLEIA (µg/L)	Spiked concentration (µg/kg)	LC-MS/MS (µg/kg)	CLEIA (µg/kg)
3.0	2.7	4.5	15	12.5	14.8
3.0	3.0	4.1	15	13.6	19.2
3.0	3.5	2.9	15	14.7	13.4
3.0	3.4	2.6	15	13.6	16.9
3.0	2.6	3.9	15	12.1	17.9
3.0	3.4	4.3	15	14.3	12.6
5.0	5.4	6.1	30	25.1	26.3
5.0	5.1	4.9	30	26.4	22.7
5.0	5.6	6.5	30	25.6	20.4
5.0	4.3	6.9	30	27.5	23.1
5.0	4.4	6.1	30	28.7	25.8
5.0	4.6	5.3	30	31.7	30.5
10	10.5	12.3	45	39.3	32.7
10	11.2	13.2	45	43.4	38.7
10	9.3	11.7	45	40.3	36.8
10	10.4	12.2	45	41.9	43.2
10	9.5	10.3	45	42.6	41.7
10	9.1	8.9	45	37.7	39.8