In order to confirm whether 7-aminonitrazepam (7-ANZP) had been obtained, the product obtaining by reducing process of nitrazepam and subsequent preliminary purification was separated by chromatographic solution hexane/acetic acetate (1:1, v/v) on silica TLC plate. Two main bands were obtained, whose R_f values were 0.72 and 0.35, respectively. The band with R_f = 0.72 was mainly nitrazepam, while another band with R_f = 0.35 was 7-ANZP. HPLC-ESI-MS was also utilized to confirm the structure of 7-ANZP (Figure 1). A prominent ion peak (M + 1, m/z 252) of 7-ANZP molecule could be clearly observed by HPLC-MS analyzing (Figure 1).

On the UV-vis spectrum of ANZP-OVA conjugate, an absorbance peak was observed at 355 nm, which is one of the characteristic peaks of 7-ANZP. An absorbance maximum was also found at 245 nm along with a smaller peak at 280 nm, which integrated the characteristic peak (245 nm) of 7-ANZP and the characteristic peak (280 nm) of OVA (Figure 2). These changes on UV-vis spectra showed that the conjugate, ANZP-OVA, could be used for further ELISA development.

A novel enzyme tracer was prepared by conjugating IgG-HRP with GNPs. The initial maximum absorbance peak of the GNPs at 520 nm was shifted to 529 nm after conjugation with IgG-HRP molecules (Figure 3), which can be expected based on the local surface plasmon resonance phenomenon [13]. This size expansion indicated the successful conjugation of the GNPs and IgG-HRP. It was important to note that the GNPs -IgG-HRP conjugate was also mono-disperse (data not shown), which will contribute to the reproducibility of the GNPs-based ELISA method.

For application of the GNPs-IgG-HRP conjugate in ELISA format, the concentrations of coating antigen, anti-7-ANZP antibody and the GNPs-IgG-HRP conjugate firstly were selected by the checkerboard method. Blocking buffer containing proteins or polymers (ovalbumin, gelatin from pork skin, polyvinyl pyrrolidone (PVP, average mw 40 kDa, PEG-10000) were also compared to obtain highest signal to noise ratio. Blocking buffer containing gelatin (0.1%) was chosen (data not shown). Under optimized concentrations of coating antigen, anti-7-ANZP antibody and the GNPs -IgG-HRP conjugate, the sensitivity (defined as the concentration when A/A₀% was 90%) could reach 5.6 pg/mL, which was nearly 20 times more sensitive than that of the traditional ELISA for 7-ANZP (Figure 4). The linear range of this nano-ELISA was 0.01~4 ng/mL (the linear equation was y = −24.338 × lgx + 39.608, R² > 99%). These results demonstrated multiple IgG-HRP molecules loading onto the surface of GNPs had endowed the conjugate producing signal amplification effect. The signal amplification effect in this method was close to that of another GNPs-based ELISA for heavy ion [19]. Note that the performance of this nano-enhanced ELISA can be improved further through more elaborate design, for example, optimizing the size of GNPs, adopting other kinds of bigger nanoparticles, or directly conjugating IgG and HRP to improve the loading of HRP molecules per GNP.

Intra-assay and inter-assay precision of the ELISA method were studied. Intra-assay precision was investigated by evaluating the variation of determination of 7-ANZP from well to well (n = 5) in the same microplate and inter-assay precision was calculated from measurements in several microplates (n = 3). The maximum coefficient of variation (CV) of intra-assay was 4.7%, while that of inter-assay was 4.6% (Table 1).

To investigate whether this nano-enhanced ELISA method could be used in real samples, the matrix effect of human urine was first tested. Considering many metabolites of benzodiazepines are excreted in urine as glucuronide conjugates, we chose β-glucuronidase to gently hydrolyze the possible glucuronide conjugates of 7-ANZP [20]. This enzymatic hydrolysis can potentially be replaced by faster acid hydrolysis.

Pretreated control human urine samples were utilized to prepare 7-ANZP standard solutions for this method. The differences of average values measured were less than 15% in the linear range from 0.01 to 4.0 ng/mL when comparing ELISA1 with ELISA2. ELISA1 was carried out with PBS buffer while ELISA2 used standards using negative urine sample matrix, which was enzymatically hydrolyzed and then diluted 5 fold by PBS buffer. The standard curve obtained using this diluted enzymatically-treated negative control as matrix was almost identical to the curve obtained using assay buffer alone (Figure 4). These results showed the matrix effect may be eliminated almost completely after enzymatic hydrolysis and appropriate dilution.

To evaluate the accuracy and precision of this novel ELISA method, the spiked human urine sample were tested by the established sample pretreatment protocol. After measuring 10 blank samples, the LOD for 7-ANZP in human urine, which is defined as the mean value of these samples plus three standard deviations of their measured signal values, was found to be 0.18 ng/mL. When detecting the urine samples spiked with 0.5, 1.5 and 4 ng/mL 7-ANZP, recoveries ranging from 83.4 to 95.5% were obtained and the relative standard deviations (RSD) were less than 11% (Table 2). These results demonstrated this nano-enhanced ELISA was reliable for 7-ANZP detection in human urine.

In order to verify the applicability of this method in a real situation, the nano-enhanced ELISA method was used in analyzing 3 positive urine samples from patients. The 7-ANZP values of positive samples were found to be 0.58, 0.89 and 2.12 ng/mL, respectively. These urine samples were further confirmed by LC-MS-MS method and the values obtained were 0.51, 0.78 and 1.95 ng/mL, respectively. It seemed the values obtained by this ELISA were all higher than that confirmed by LC-MS-MS method, but the differences were all less than 14%. Considering all the values were at ppb level and the samples were pretreated by a simple way, this difference should be acceptable.

Polycolonal rabbit antibody against 7-aminonitrazepam (7-ANZP) was produced in our lab. 7-aminonitrazepam (7-ANZP) was purchased from National Institutes for food and drug Control (NIFDC, Beijing, China). Ovalbumin (OVA), 3,3′,5,5′-tetramethylbenzidine (TMB), chloroauric acid (HAuCl₄) and trisodium citrate were purchased from Sigma-Aldrich (St. Louis, MO, USA). The peroxidase-conjugated goat anti-mouse IgG (IgG-HRP) was purchased from Kangchen Biotechnology Company (Shanghai, China). Microtiter plates were obtained from Costar Group, Inc., (Bethesda, MD, USA). Other chemical regents were all analytical grade and from Shanghai Chemical Reagents Company (Shanghai, China). All buffers used in the experiments were prepared using Milli-Q water from Milli-Q system (Millipore, Bedford, MA, USA).

Coating buffer, 50 mmol/L carbonate buffer (pH 9.5); dilution buffer (phosphate buffered saline (PBS)), 10 mmol/L sodium phosphate buffer (pH 7.4) containing 140 mmol/L NaCl; washing buffer, PBS containing 0.05% (v/v) Tween 20 (PBST); TMB solution, 50 mmol/L sodium citrate buffer (pH 5.0) containing 0.01% (w/v) TMB and 0.005% (v/v) H₂O₂.

The hapten of 7-ANZP and protein conjugate were prepared according to the reference in which 7-aminochlonazepam, an analogue of 7-ANZP, was prepared [4]. Briefly, 1.68 g iron powder and 200 mg ammonium chloride were suspended in 5 mL water and heated for 15 min, after which 2.8 g nitrazepam in 100 mL methanol was added to the mixture. After refluxing for 5 h, iron powder was removed by filtration and the methanol was evaporated in vacuum. The precipitate was collected and recrystallized with methanol to give 2.24 g (2.77 g) of fawn crystalloid.

The 7-ANZP-protein conjugate was prepared according to the diazotization method. 10 mg 7-aminonitrazepam was dissolved in warm 0.05 M sulfuric acid (4 mL, 70 °C). The solution was then cooled in an ice bath, 1 mL of freshly prepared sodium nitrate (19 mg/mL) was added dropwise within 3 min and stirred for 30 min. An OVA solution (100 mg in 4 mL of 0.5 M borate, pH 9.4) was cooled down to 0 °C, and the diazotized 7-ANZP solution was added to the cooled OVA solution within 15 min. The pH of the solution was maintained between 9.0 and 9.5 by adding sodium hydroxide (1 M). After 4 h, the reaction mixture was stood at room temperature, exhaustively dialyzed against 10 mM phosphate buffer (pH 7.2, 9 × 2 L) at 4 °C and stored at −20 °C prior to use.

Gold nanoparticles (20 nm) were prepared by reducing HAuCl4 with trisodium citrate. Flasks and magnetic stir bars used were cleaned sufficiently with aqua regia. 100 mL of 0.1% HAuCl₄ solution was boiled and then vigorously stirred in round-bottom flask. Then 2 mL of 1% trisodium citrate solution was added quickly to the solution. After a color change from pale yellow to dark red, which indicated the formation of GNPs, the solution was maintained for another 10 min at boiling temperature and then stirred to cool down.

The GNPs-IgG-HRP conjugate was prepared as follow. Exactly 1 mL of GNP (20 nm) solution was mixed with 1, 2 or 3 μL of K₂CO₃ (200 mM), and then 100 μL of IgG-HRP solution (100 μg/mL) was added to 1 mL of each of the treated GNP solution. Each mixture was stirred for 30 min and then centrifuged at 15,000 × g for 15 min at 4 °C to remove the excess of antibody. The clear supernatant was carefully removed, and the precipitated GNPs conjugates were resuspended in 1 mL of PBS buffer (0.01 M, pH 7.4) and stored at 4 °C. The conjugates prepared through the addition of different amounts of K₂CO₃ were evaluated by adding 100 uL of NaCl (10%) and observing their color change. After 24 h at 4 °C, the conjugates prepared with 1 or 2 μL of K₂CO₃ appeared blue to some extent, while the conjugates prepared with 3 uL of K₂CO₃ still appeared red. Finally, this conjugate, prepared by adding 3 μL of K₂CO₃, was chosen for further use. The UV-vis spectroscopy of the conjugate was analyzed to evaluate the size change.

The procedure of the nano-enhanced ELISA was almost the same as that of a traditional indirect ELISA method except that the IgG-HRP was replaced with the GNPs-IgG-HRP conjugate. The general procedure developed in the assay was as follows. 100 μL of 7-ANZP-OVA (15 μg/mL) was added to each well of a 96-well microplate and immobilized at 37 °C for 2 h. Coating antigen was decanted and microplates were washed by PBST. The microplates were then blocked by incubating with 200 μL of blocking buffer (1% BSA in PBST) for 2 h at room temperature. After decanting the blocking buffer and washing the microplates, 50 μL of 7-ANZP standard solutions, or test samples, and 50 μL of anti-7-ANZP antibody solution in PBST buffer containing 0.1% gelatin were added to the wells. The microplates were then incubated at 37 °C for 30 min. The reaction mixtures in wells were then decanted and the wells were washed, then 100 μL of diluted GNPs-IgG-HRP conjugate was added to the each well. After incubation for 30 min, the wells were washed again. HRP substrate containing TMB and H₂O₂ solutions was added and color was developed for 15 min. Then the reactions were stopped by addition of 2 M sulfuric acid (100 μL) to each well. The optical density was read at 450 nm with a microplate reader within 5 min after stopping the reaction.

Blank urine samples were obtained from healthy individuals as negative samples. Urine samples, collected from 3 patients at the Fourth Hospital in Wuxi who were known to be receiving oral doses of clonazepams (CZP), were analyzed as positive samples. The experiment was approved by the local ethics committee of the hospital.

The urine samples were filtered through Whatman no. 1 filter paper, and stored frozen (−20 °C) in aliquots of 10 mL until required for analysis. Aliquot of 1.0 mL urine sample was centrifuged at 5000× g for 10 min at room temperature and to 0.5 mL of the supernatant 0.5 mL of β-glucuronidase (6000 units) was added and incubated for 2.5 h to achieve hydrolysis of the conjugates. Then the hydrolysate was diluted further with phosphate buffer (0.1 M, pH 7.4) for analysis. For validation of the assay, 7-ANZP standard solution (0.1 mg/mL) was added to the negative urine sample in order to obtain 7-ANZP spiked samples. After standing for 30 min, the spiked samples were centrifuged, hydrolyzed and diluted as the above method.