Research article

Received: 3 February 2013, Revised: 10 April 2013, Accepted: 7 May 2013 Published online in Wiley Online Library: 20 June 2013

(wileyonlinelibrary.com) DOI 10.1002/bio.2544

Development of a highly sensitivechemiluminescence enzyme immunoassayusing enhanced luminol as substrateXiaoqi Tao,a Wenjun Wang,b Zhanhui Wang,a Xingyuan Cao,a Jinghui Zhu,a

Lanlan Niu,b Xiaoping Wu,b Haiyang Jianga* and Jianzhong Shena*

ABSTRACT: In this study, a high sensitivity chemiluminescence enzyme immunoassay (CLEIA) based on novel enhancerswas developed. Under optimal conditions, we developed an enhanced chemiluminescence reaction (ECR) catalyzed byhorseradish peroxidase (HRP-C) in the presence of 3-(10’-phenothiazinyl) propane-1-sulfonate (SPTZ) and 4-morpholinopyridine(MORP) as enhancers. The limit of detection of the newly prepared chemiluminescent cocktail for HRP was 0.33pg/well, whichis lower than that of commercial Super Signal substrate. The results showed that this novel chemiluminescent cocktail cansignificantly increase the light output of HRP-catalyzed ECR, which can be translated into a corresponding improvement insensitivity. Similar improvements were observed in CLEIA for the determination of chloramphenicol in milk. In addition, the ECR ofN-azoles as secondary enhancer was also presented. Copyright © 2013 John Wiley & Sons, Ltd.

Keywords: 3-(10’-phenothiazinyl) propane-1-sulfonate; 4-morpholinopyridine; horseradish peroxidase; chemiluminescence enzymeimmunoassay; chloramphenicol

* Correspondence to: Jianzhong Shen, Department of Pharmacology andToxicology, College of Veterinary Medicine, China Agricultural University,Yuan Ming Yuan West Road NO.2, Beijing 100193, China. Tel: +86 01062732803; Fax: +86 010 62731032. E-mail: sjz@cau.edu.cn

Haiyang Jiang, Department of Pharmacology and Toxicology, College ofVeterinary Medicine, China Agricultural University, Yuan Ming Yuan WestRoad NO.2, Beijing 100193, China. Tel: +86 010 62732802; Fax: +86 01062731032. E-mail: haiyang@cau.edu.cn

Xiaoqi Tao and Wenjun Wang contributed equally to this work.

a Department of Pharmacology and Toxicology, College of VeterinaryMedicine, China Agricultural University, Beijing100193, China

b Beijing WDWK Biotech Co., Ltd., Beijing100085, China

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IntroductionThe horseradish peroxidase (HRP)-catalyzed chemiluminescentoxidation of luminol is used widely in bioanalytical methods,such as western blots, immunohistochemistry and chemilumi-nescent enzyme immunoassays (CLEIA). A chemiluminescence(CL) assay is often more sensitive than other methods (1,2) andmuch effort has been made to further improve its efficiencyand analytical performance. In particular, the addition ofenhancers to the reaction substrate greatly increases lightoutput and duration kinetics (3). Luminol oxidation leads tothe formation of a 3-aminophthalate ion in an excited state,which emits light on returning to the ground state (Fig. 1,reaction 10). The emission spectrum shows a maximumwavelength at 425 nm (4). The mechanism of the enhancedchemiluminescence reaction (ECR) has been described inprevious reports (5,6), in which luminol and an enhancer wereoxidized simultaneously. During the first step of ECR, theenhancer molecule, which is a better substrate for HRP thanluminol, is oxidized by hydrogen peroxide in the presence ofHRP according to the ‘ping-pong’ mechanism (Fig. 1).

Several analytes have been studied as enhancers for theluminol–peroxidase system, including phenolic and aminederivatives (7–9), indophenols (10), 4-phenylylboronic acid (11),4-methoxyphenol, 4-hydroxy-biphenyl, 4-(1H-pyrrol-1-yl)-phenol(12) and different polymers (13). Use of 3-(10’-phenothiazinyl)propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORP)(Fig. 2) as primary and secondary enhancers, respectively,allowed the development of a sensitive CL method for thedetermination of different plant peroxidases (14–17). Subsequently,ECR with the above-mentioned enhancers was successfully usedin the development of ultrasensitive immunochemical methodsfor the determination of human thyroglobulin, ochratoxin A and

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bacterium Yersinia enterocolitica (16,18,19). Although previousstudies optimized the ECR conditions and obtained good sensitivity(14–17), follow-up stability, significant for a mature product, wasnot determined.Chloramphenicol (CAP) is an effective broad-spectrum

antibiotic which was widely used in both human and veterinarypractice for the prevention and treatment of many bacterialinfections. However, CAP is a hemotoxic substance for humansand can cause bone marrow depression, aplastic anemia andacute leukemia (20). These potential hazards led to a prohibitionon its use in food-producing animals in many countries,including China, the USA and the EU (21,22). However, CAP is stillillegally used as an antibiotic in animal husbandry because of itslow cost and excellent antibacterial effect. Therefore, there is anurgent need to develop a rapid and sensitive method for thedetermination of CAP at trace levels in animal-derived food. In aprevious study, we developed a competitive direct chemiluminescent

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Figure 1. HRP catalyzed the oxidation of luminol by the ‘ping-pong’ mechanismwith the enhancer.

Figure 2. Chemical structures of 3-(10ʹ-phenothiazinyl)ropane-1-sulfonate (SPTZ)(a) and 4-morpholinopyridine (MORP) (b).

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enzyme-liked immunosorbent assay (CL-ELISA) for detecting CAPresidues in milk, milk powder, honey, eggs and chicken muscle usingthe SuperSignal CL substrate (23).

Under optimized conditions, a highly sensitive CLEIA for thedetermination of CAP in milk was developed. This was basedon the enzymatic oxidation of luminol by sodium peroxide inthe presence of SPTZ and MORP as enhancers and HRP-C asthe biocatalyst. The obtained results (low detection limit ofCAP, good stability, etc.) demonstrated that this establishedmethod gave a significant improvement in the sensitivity of CLdetermination of HRP-C activity.

Materials and methods

Apparatus

The CL reader, Veritas Microplate Luminometer was from TurnerBioSystems (Sunnyvale, CA, USA). White opaque microplates (‘highbinding’grade; Costar,Washington, DC, USA)were used for the CLEIAassay together with a Milli-Q system (Millipore, Billerica, MA, USA).

Reagents

Standards were as follows: CAP (99% purity, Sigma Aldrich, St.Louis, MO, USA); luminol sodium (98.3% purity), SPTZ (98.0%purity) and MORP (97% purity) were purchased from SeebioBiotech, Inc (Shanghai, China). HRP (RZ 3.0) was purchasedfrom Sigma. Imidazole (99% purity), 1,2,3-triazole (99% purity),1,2,4-triazole (99% purity), 1-methylimidazole (99% purity),

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imidazole (99% purity), sodium perborate (NaBO3, 98% purity)and 30% H2O2 were purchased from Aladdin (Shanghai, China).All other compounds were obtained from commercial sourcesand used without further purification. Other chemicals andorganic solvents were of reagent grade and were from BeijingChemical Co. (Beijing, China).

The SuperSignal CL substrate solution was purchased fromPierce (Rockford, IL, USA).

The polyclonal anti-CAP serum (PAb) antibody was obtainedfrom WDWK Biotech Co. (Beijing, China).

Buffers

The following buffers and solutions were used in CLEIA assay:(a) coating buffer (CB, pH 9.6) – 0.05M carbonate buffer, madewith 1.59 g Na2CO3 and 2.93 g NaHCO3 in 1 L of purified water;(b) blocking buffer – 0.01M PBS containing 0.5% casein;(c) washing solution (PBST) – 0.01M PBS containing 0.05%Tween-20; (d) 0.2M sodium phosphate solution (pH 7.2)containing 11.0 g NaH2PO4 � 2H2O, 51.6 g Na2HPO4 � 12H2O in1 L of purified water; (e) PBS (pH 7.4) – 0.01M PBS was preparedby dissolving 8.0 g NaCl, 0.2 g KCl, 0.24 g KH2PO4 and 3.63 gNa2HPO4 � 12H2O in 1 L of purified water; (f) solution A – 0.36MK4Fe(CN)6 � 3H2O, solution B – 1.04M ZnSO4 � 7H2O. All bufferswere prepared using MilliQ H2O (18 MΩ/cm).

Catalytic luminol oxidation condition

Catalytic luminol oxidationwas assayed as follows: 0.05–0.3mmol/Lluminol, 0.9–3.1mmol/L SPTZ, 0.5–25.0mmol/L MORP and1.0–3.0mmol/L sodium peroxide (both reagents were prelim-inarily dissolved in Tris buffer at the relevant pH and concen-tration) were prepared with 10–100mmol/L Tris, pH 8.0–9.0for the enzyme immunoassay. The enzymatic reaction wasinitiated by adding 10 mL of peroxidase solution (2 mg/mL)dissolved in Tris at relevant pH and concentration. CL kineticswas measured at 425 nm with a CL reader, 3min after theaddition of the substrate at room temperature; the resultswere expressed in relative light units (RLU). The CL signalformed in the absence of enzyme was used as a control.

Effect of nucleophilic acylation and N-azoles catalysts onSPTZ-enhanced substrates

The working solution was freshly prepared with the followingconcentrations: 0.17mM luminol sodium salt, 2.1mM SPTZ and2mM sodium perborate in 0.10M Tris (pH 8.5). This solutionwas split into portions, and the following compounds wereadded to each portion to a series concentration: (a) MORP(0.5–25.0mM), (b) 1,2,3-triazole (3–85mM), (c) 1,2,4-triazole(1.5–85mM), (d) imidazole (1.5–75mM), (e) 1-methylimidazole(1.5–75mM), and (f) reference (without enhancer). The enzymeaddition protocol described above was used.

Light emission from working solutions containing MORP:effect of pH

Working solutions of MORP were freshly prepared. The MORPworking solution contained 0.17mM luminol sodium salt,2.1mM SPTZ, 1.5mM MORP and 2mM sodium perborate in0.10M Tris (pH 8.5). The pH for each working solution wasadjusted with negligible amounts of HCl (1M) or NaOH (1M) in

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A high sensitivity CLEIA based on novelty enhancers

the interval pH 8–9. The enzyme addition protocol describedabove was used.

Standard curve for HRP

Ten microliters of HRP diluted in Tris buffer (0.1M, pH 8.5) in therange 0–100 pg/well were analyzed in a microplate usingalternatively NoMORP, MORP and SuperSignal working solutions(100 mL) prepared as described above.

Application of the MORP–SPTZ–luminol CL reaction in adirect competitive CLEIA

A direct competitive CLEIA for CAP previously developed inour laboratory (23) was used to evaluate the analyticalperformance of the new CL cocktail. Briefly, high-bindingwhite plates were coated overnight at 4 �C with 100 mL ofthe polyclonal anti-CAP serum (PAb) dissolved in buffer a(1.5 mg/mL). The plates were washed with 260 mL/well of bufferc manually three times, blocked with 150 mL/well of buffer band incubated at 37 �C for 1 h. After the plates were washedas described above (conditioned ELISA plates can be storedat 4 �C for one week), then 80 mL/well of standard in buffer dor sample solution, followed by 20 mL/well of HRP-conjugatedCAP at a dilution of 1/160 000 in buffer d were added, respec-tively. The competitive reaction took place for 15min at roomtemperature. After washing five times, the HRP tracer activitywas revealed by adding 100 mL/well of a freshly preparedsubstrate mixture of SuperSignal substrate solution or MORP–SPTZ–luminol CL cocktail reaction. The intensity of lightemission was measured at 425 nm with a CL reader, 3min afteraddition of the substrate and the results were expressed in RLU.Calibration curves were obtained by plotting B/B0 againstthe logarithm of analyte concentration and fitted to a four-parameter logistic equation using Origin (v. 8.0; Microcal,Northampton, MA, USA) software packages.

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Milk sample preparation

For extraction of CAP from milk, 500mL of solution A and and500mL of solution B were added to 10mL of milk, mixed thor-oughly and then centrifuged for 10min at 4000g and 4 �C. Analiquot (4.4mL) of aqueous supernatant (amount to 4mL milk)was thoroughly mixed with 8.0mL of ethyl acetate for 10minin a new tube. Following centrifugation at 4000 g for 10min,4mL of organic supernatant (amount to 2mL milk) wastransferred to a new tube and dried by nitrogen at 60 �C. Theresidue was dissolved in 2mL of buffer d. The sample solutionwas used for determination.

Results and discussion

‘Ping-pong’ mechanism

Use of SPTZ and MORP as primary and secondary enhancers,respectively, allowed the development of the sensitivechemiluminescent method for the determination of horseradishperoxidase; which catalyzed the oxidation of luminol by the‘ping-pong’ mechanism (Fig. 1). According to the mechanism,the native Fe(III) enzyme (HRP) is oxidized by peroxide to HRP-Iin a two-electron oxidation (Fig. 1, reaction 1). HRP-I then returnsto its native state, HRP, by reaction with the primary enhancer (E)

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or luminol anion (LH–) in two one-electron transfer steps,with HRP-II as an intermediate (Fig. 1, reactions 2 and 4).Subsequently, HRP-II oxidized another primary enhancer (E) orluminol anion (LH–), returning to its native state in which it willparticipate in another oxidization cycle (Fig. 1, reactions 3 and5). In each of these steps, the primary enhancer (E) or luminolanion (LH–) is oxidized to its radical form (E•). The reaction ofluminol with HRP-II is ~ 100-fold less rapid than the reaction ofluminol with HRP-I. Luminescence enhancers react more rapidlywith HRP-II than luminol does, thereby accelerating enzymeturnover. In the next step, the enhancer radical E oxidizesluminol anion LH– to the key intermediate L (Fig. 1, reaction 6).Dismutation of L regenerates LH– while producing atwo-electron oxidized luminol species, a diazaquinone (L) (Fig. 1,reaction 7). The diazaquinone is then attacked by hydrogenperoxide anion HO2

– (Fig. 1, reaction 8). An intermediateperoxide species is formed and then collapses, with loss ofnitrogen, to 3-aminophthalate in its excited state, AP* (Fig. 1,reaction 9). The decay of AP* to aminophthalate, AP, isresponsible for the chemiluminescent light emission at 425nm(Fig. 1, reaction 9).

Optimization of catalytic luminol oxidation

In order to get the lowest value of the lower detection limit (LDL)(i.e. the HRP concentration required for CL to be twice that of thesame solution without HRP), concentrations of luminol, sodiumperoxide, SPTZ and Tris as well as the pH of the reaction mixturewere optimized. In each case, the ratios of peroxidase-catalyzedCL to background were determined (Figs 3,4). The concentrationof MORP was also optimized (Fig. 5). The most favorableconditions for HRP were 100mM Tris buffer (pH 8.5) containing0.17mM luminol, 2mM NaBO3, 2.1mM SPTZ and 1.5mM MORP.In the presence of SPTZ combined with MORP, HRP-induced CLreached a maximum value at 3min after the initiation of luminoloxidation and then decreased slowly (Fig. 6).Actually, signal enhancement for MORP is quite strong even at

very low concentrations. When the concentration is 1.5mM, thevalue of CL reaches a maximum. There is a sudden decrease inCL signal intensity with a further increase in the concentrationof MORP. By contrast, N-azoles either decrease their enhancedeffect slowly (imidazole) or reach a stable level at a very highconcentration (1-methylimidazole, 1,2,3-triazole, 1,2,4-triazole)(Fig. 5). Based on the above results, the use of N-azoles assecondary enhancers provides a considerable adjustmentfor the CL light output, which could not be achieved using4-aminopyridines as a secondary enhancer (MORP) (Fig. 5).Therefore, it is possible to tightly regulate the initial light

signal and its duration through the use of N-azoles as secondaryenhancers. This feature is highly valuable because it allows fineadjustment of the chemiluminescent HRP substrate for specificpurposes. For example, it can be used to obtain a moremoderate enhancement effect and much longer signal duration,in order to maximize the amount of light generated in a givenexposure window. The reaction mechanism of N-azoles assecondary enhancers may share some similarities with that of4-aminopyridines. For example, they both attack dizaquinoneL, thereby generating more reactive intermediates for theperoxide. The differences in the signal enhancement effectbetween the N-azoles and 4-aminopyridines may be due to theirdifferent affinity.

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Figure 3. Effect of luminol (a), SPTZ (b) and concentration of sodium peroxide (c) and Tris buffer (d) on the ratio of HRP-induced CL to background. Conditions: [HRP] = 0.45pM, [MORP] = 1.5mM, pH 8.5; (a) [NaBO3] = 2mM, [SPTZ] = 2.1mM and 100mM Tris buffer; (b) [NaBO3] = 2mM, [luminol] = 0.17mM and 100mM Tris buffer; (c) [luminol] =0.17mM, [SPTZ] = 2.1mM and 100mM Tris buffer; (d) [NaBO3] = 2mM, [luminol] = 0.17mM, [SPTZ] = 2.1mM. CL intensity was recorded 3min after the start of the reaction.Each point represents the mean of triplicate experiments. Vertical bars indicate� SD about the mean.

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Thermal stability

In order to ensure long-term stability, the CL substrate compo-nent was prepared as two separated solutions (A and B), whichcan be mixed in equal amounts to produce the working solutionwhen required. CL substrate solutions A and B were dispensedinto multiple portions, and stored at 4 and 37 �C for 1, 2, 5, 6,7, 8, 9 and 10 days. S/N, representing the ratio of the CL value

Figure 4. Working solution pH optimal value for a MORP-catalyzed system, withthe working solution based on luminol (0.17mM)/SPTZ (2.1mM)/NaBO3 (2mM)/MORP (0.15mM)/Tris buffer (0.1M, pH 8.5)/HRP (0.45 pM). Each point is calculatedintegrating the signal during the first 3-min run of a kinetic measurement.

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in HRP (2 mg/mL) to that in PBS, was applied to assess thesolution stability of individual storage conditions. S/N at 37 �Cwas almost identical to at 4 �C from day 0 to day 10 (Fig. 7). Thismeans that the CL solution can be stably preserved for 1 year at4 �C, and used in kits for various purposes to detect HRP.

Sensitivity

The detectability of the enzyme label is one of the most impor-tant factors determining the detection limit of assays, providing

Figure 5. The luminol/SPTZ/NaBO3/HRP system’s dependence on the concentra-tion of secondary enhancer: (■) MORP; (●) 1,2,3-triazole; (▲) 1,2,4-triazole; (▼)imidazole; (◀) 1-methylimidazole; (▶) without enhancer.

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Figure 6. Kinetic measurement of light output intensity from luminol (0.17mM)/SPTZ (2.1mM)/NaBO3 (2mM)/MORP (0.15mM)/Tris buffer (0.1M, pH 8.5)/HRP (0.45pM) systems.

Figure 7. Thermal stability. (■) Solutions A and B were stored at 4 �C on variousdays; (●) solutions A and B were stored at 37 �C on various days.

Figure 8. Comparison of HRP dose–response curves for a MORP-catalyzed systemand a noncatalyzed system, both having a working solution based on luminol(0.17mM)/SPTZ (2.1mM)/NaBO3 (2mM)/Tris buffer (0.1M, pH 8.5) and SuperSignalsubstrate: (▲) NoMORP (no catalyst added); (●) SuperSignal substrate (■) MORP(1.5mM). Each point is calculated by integrating the signal during the first 3-minrun of a kinetic measurement. Data are the means of triplicate assays with theblank subtracted. Error bars: � 1 SD.

Figure 9. Normalized standard curve by CLEIA for CAP with MORP-catalyzedchemiluminescent cocktail (■) compared with the standard curve obtained bySuperSignal substrate (●).

A high sensitivity CLEIA based on novelty enhancers

that a low degree of non-specific binding of the detectionreagents occurs (24). Comparison of the dose–response curvefor HRP using the SPTZ-enhanced luminol/peroxide substratewith MORP and SuperSignal is shown in Fig. 8. The detectionlimit for HRP was 0.33 pg/well for the substrate containingMORP. Compared with 1.0 pg/well for the SuperSignal substrateand 10 pg/well for the NoMORP substrate, this indicates that theproposed CL solution in this study was more sensitive thanSuperSignal CL substrate.

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Application of the MORP–SPTZ–luminol CL cocktail in adirect competitive ELISA

A direct competitive CLEIA for the determination of CAP wascarried out to compare the performance of the new chemilumi-nescent cocktail with SuperSignal substrate. Calibration curvesobtained by plotting the B/B0 against the logarithm of CAP

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concentration (Fig. 9) ,showed that the detection limit ofcocktail containing MORP for CAP was ~ 2.7 times lower thanthat obtained with the SuperSignal substrate, thereby providinga steeper calibration curve. The MORP reagent allowed reachinga limit of detection (LOD) of 0.6 ng/L, compared with that ofthe SuperSignal substrate cocktail, which reached a LOD of1.6 ng/L. The linear range extended to 1.37–17.2 ng/L for MORPand 3.4–55.0 ng/L for SuperSignal substrate.

Recovery and precision

Milk samples were spiked with CAP at 2, 8 and 16 ng/L and thenanalyzed with the CL-ELISA. Each sample was evaluated sixtimes in duplicate and on three different days to verify therepeatability of the assay.In sample analysis, the intra-assay recoveries were in the

range 90.0–101.2% (Table 1). The inter-assay recoveries werein the range 75.0–95.0%. The coefficients of variation (CVs)

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Table 1. Intra- and interassay variations in milk spiked with CAP.

Drug Level(ng/L)

Intra-assaya Inter-assayb

Measured Recovery Measured Recovery

(ng/L) (%) (ng/L) (%)

CAP 2 1.8� 0.2 90.0 1.5� 0.2 75.08 8.1� 0.5 101.2 7.5� 0.6 93.816 14.8� 1.6 92.5 15.2� 1.7 95.0

aIntra-assay variation was determined using six replicates on a single day.bInter-assay variation was determined using six replicates on three different days.

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were all< 15%. In general, the direct competitive CLEIAwith the newchemiluminescent cocktail showed good results in milk and laid thefoundation for the commercialization of CL-ELSA kits for CAP.

ConclusionThe incorporation of an acylation catalyst in enhancer/luminol/oxidant HRP substrates is highly advantageous, especially in thecase of 4-aminopyridine enhancers. Although the exact mode ofaction of acylation catalysts has not yet been fully investigated, itis clear that the very significant increase in light output observedin their presence can be translated into a corresponding improve-ment in sensitivity of chemiluminescent assays. Moreover, thehigh stability and sensitivity of the new chemiluminescent cocktailwere verified, and could be applied in the future CL detection kits.However, the mechanism of N-azoles as secondary enhancersneeds to be further explored.

Acknowledgements

This study was supported by Chinese Universities Scientific Fund(2013QJ047) and Technology Pillar Program in the TwentiethFive-Year Plan Period (2011BAK10B01, 2011BAZ0319816).

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