A design of reaction-controlled chemiluminescence imaging and its application

Analytica Chimica Acta 543 (2005) 222–228

A design of reaction-controlled chemiluminescence imaging and its application Lirong Luo b , Zhujun Zhang a,∗ , Lifeng Ma a a

Department of Chemistry, Shaanxi Normal University, South Chang’an Road Number 199, Xi’an, Shaanxi 710062, China b Department of Chemistry, Southwest Normal University, ChongQing 400715, China Received 22 January 2005; received in revised form 23 March 2005; accepted 29 March 2005 Available online 10 May 2005

Abstract The paper presented a design of reaction-controlled chemiluminescence (CL) imaging analysis method. The design was based on controlling pH to initiate CL reaction and obtain high sensitivity. The pH value of CL reaction was controlled by ammonia, which was produced by injecting NaOH solution to NH4 Cl solution, and the amount of ammonia could be adjusted by varying concentration, pumping time and flow rate of NaOH solution or varying concentration of NH4 Cl solution. The pH of CL reagents in 96-well microtiter plates increased continuously from the same initial value due to the absorbance of ammonia and the relative CL intensity increased with the increasing pH. Based on above reaction-controlled design, the CL reaction in 96 wells could be initiated at the same time and the total CL signal of each well could be monitored. As results of above operation, a high sensitivity and better reproducibility could be obtained. A luminol, H2 O2 , CL system for determination of hemoglobin (Hb) was selected to validate the presented design. The CL intensity was proportional with the concentration of Hb in the range of 1.0 × 10−9 to 1.0 × 10−7 mol l−1 and the detection limit was 3.0 × 10−10 mol l−1 (3σ), the relative standard deviation (R.S.D.) for 11 parallel measurements of 1.0 × 10−8 mol l−1 Hb was 2.7%. © 2005 Elsevier B.V. All rights reserved. Keywords: Chemiluminescence; Imaging; Design; Application

1. Introduction Luminescence image technologies have shown progress in recent years [1–6]. Among detection modes for luminescence image, chemiluminescence (CL) analysis offers a high sensitivity, wide linear range and simple instrument and has been employed to design imaging systems [7–15]. However, CL imaging is susceptible to several problems. Firstly, the CL reaction must occur with glow-type light kinetics, which permits easy handling and standardization of the experimental conditions because of lag time between initiation of the reaction and data collection. Most flash-type CL reactions are unsuitable for sensitive and reproducible determinations in CL imaging assay. In order to solve this problem, fastemitting (flash-type) CL reaction is tuned to furnish a slower∗

Corresponding author. Tel.: +86 29 85308748; fax: +86 29 85307774. E-mail address: [email protected] (Z. Zhang).

0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2005.03.073

emitting (glow-type) process that is suitable for simple CL imaging [16,17]. Typically, the CL reaction of luminol-H2 O2 horseradish peroxidase (HRP) system is a flash-type reaction and is unsuitable for simple CL imaging assay. The reaction can be tuned to glow-type reaction by using p-iodophenol (p-IP) as enhancer [18–20]. However, it is difficult to tune most flash-type reactions to glow-type reactions. Secondly, the total CL signal cannot be monitored even some glow-type reactions are used, which will induce a relative poor sensitivity. For example, the kinetics of the enhanced luminolH2 O2 -HRP system usually shows a rapid increase until the maximum value is reached, followed by a slow decrease to the background level after approximately few minutes [21]. When CL reagents are manually added to 96- (384-) well plates or microarray systems by a pipette, it is a great challenge for the operator to add CL reagents to 96- or 384wells within few minutes [12,22]; therefore, the total CL signal cannot be collected and the maximum value will be

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missed. Moreover, a poor reproducibility of CL signal will be induced because the CL reaction cannot be initiated at the same time when CL reagents are manually added to 96(384-) well plates or microarray systems. In order to initiate the CL reaction at the same time, automated pipetting systems have been applied to CL imaging assay, CL signal with good reproducibility could be obtained. However, the instrumental spending will increase and the lag time between initiation of the reaction and data collection still exists [23–25]. Therefore, a simple design to solve these problems would be desirable. Here, we report a design of reaction-controlled CL imaging analysis method to solve above problems. The design was based on controlling pH to initiate CL reaction and obtain high sensitivity. Luminol-based CL reactions were chosen as model. It is well known that luminol emits strong chemiluminescence in alkaline medium [26,27], no CL signal was observed under the acidic conditions. Therefore, the CL reaction can be initiated and controlled by controlling pH. The pH of CL reagents in 96-well microtiter plates was adjusted to 5.0 before detection, and then the pH of CL reagents increased continuously from the same initial value due to the absorbance of ammonia which was produced by injecting NaOH solution to NH4 Cl solution. The relative CL intensity increased with the increasing pH and then decreased slowly after reaching the maximum value. In this system, the amount of ammonia could be adjusted by varying concentration, pumping time and flow rate of NaOH solution or varying concentration of NH4 Cl solution. A luminol, H2 O2 , CL system for determination of Hb was selected to validate the presented design. The CL intensity was proportional with the concentration of Hb in the range of 1.0 × 10−9 to 1.0 × 10−7 mol l−1 and the detection limit was 3.0 × 10−10 mol l−1 (3σ). Comparing with the conventional CL imaging method without pH control, the sensitivity of the proposed method has been improved over 10 times for Hb and the reproducibility was better. The reliability of the assay method was established by parallel determination and by standard-addition method (the R.S.D. for 11 parallel measurements of 1.0 × 10−8 mol l−1 Hb was 2.7%, recoveries = 94–105%). Present method has several advantages over conventional CL imaging method. Firstly, the lag time between initiation of the reaction and data collection was eliminated, the total CL signal of each well were monitored and the CL reaction in 96-wells could be initiated at the same time. As results of above operation, a high sensitivity and better reproducibility were obtained, as low as 1.0 × 10−9 mol l−1 Hb could be detected by using the proposed model. In contrast, at least 5.0 × 10−8 mol l−1 Hb was required to produce an unambiguous signal in conventional CL imaging method. Secondly, the reaction-controlled CL imaging method was expected to monitor fast-emitting CL reaction directly. Overall, experimental results showed that the reaction-controlled CL imaging detection model has potential to be used in immunoassay.

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2. Experimental 2.1. Reagents and solutions All chemicals and reagents used in this study were of analytical grade; the water used for the preparation of solutions was deionized and doubly distilled. Hemoglobin (Hb, from bovine blood) was purchased from Sigma. Tris(hydroxymethy) aminomethane was obtained from Shanghai Chemical Plant (Shanghai, China). Luminol and p-IP were obtained from Kangpei Technology Company (Xi’an, China). Hydrochloric acid, NaOH and NH4 Cl were obtained from Xi’an Chemical Plant (Xi’an, China). Assay buffer was 0.1 mol l−1 Tris–HCl solution, pH 5.0 or 8.5. A 5.0 × 10−2 mol l−1 luminol stock solution was prepared by dissolving 9.32 g luminol in 20 ml 0.1 mol l−1 NaOH solution and then dilution to 1000 ml with water. H2 O2 , luminol, p-IP working solution were prepared by appropriate dilution with 0.1 mol l−1 Tris–HCl buffer, pH 5.0 or 8.5. Standard Hb (1.0 × 10−5 mol l−1 ) solution was prepared by dissolving 0.0335 g Hb in 50 ml deionized water and stored below 4 ◦ C. 2.2. Instrumentation The schematic diagram of the reaction-controlled CL imaging employed in this work is shown in Fig. 1. Ammonia was produced in a hand-made gas chamber, the gas chamber was made of six glass slides, the up cover and the cell body were sealed with plasticene and the size of the gas chamber was 21.0 cm × 16.0 cm × 10.0 cm. A peristaltic pump (Shanghai Qingpu Huxi Analytical Instrument Plant, Shanghai, China) was used to deliver NaOH solution. Polytetrafluoroethylene (PTFE) tubing (0.8 mm i.d.) and rubber tubing (length 10.0 cm, i.d. 1.0 mm) were used as connection material in the system. Crucible (vol. 50 ml) was used to contain NH4 Cl solution; 96-well microtiter plates were placed directly under the CCD camera. The change of CL signal was

Fig. 1. Schematic diagram of CL imaging system: a, NaOH solution; b, rubber screw; c, PTFE tube; d, crucible; e, glass gas chamber; f, shelf for 96-well plates; g, detector and P, peristaltic pump.

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detected and recorded with FluorchemTM IS-8800 system (Alpha Innotech, CA, USA). Thermoelectric cooling CCD camera was used as detector, data acquisition and treatment were performed with Alpha Ease FC software running under Windows 2000. The 96-well transparent microtiter plates used for the assay were obtained from Corning Incorporation (Corning, USA). P20 adjustable air-displacement pipette was obtained from Gilson Inc. (Gilson, France). 2.3. Sample preparation The standard human Hb solution was prepared from fresh human blood. Its concentration was determined by cyanmetHb method. The working curve was obtained by properly diluting the above standard solution. Fresh blood was collected in clean plastic tubes; no anticoagulant was added to the blood samples allowing the blood coagulation naturally and then centrifuged at 3000 rpm for 5 min. The supernatant part was extracted as serum with a mini sample collector. Sera were kept for 3 days at 4 ◦ C in a refrigerator. 2.4. Procedures 2.4.1. Procedure for reaction-controlled chemiluminescence imaging The CL reagents were 50 ␮l luminol solution with p-IP, 50 ␮l H2 O2 solution and 50 ␮l Hb standards or sample, the pH of CL reagents was adjusted to 5.0 by 0.1 mol l−1 Tris–HCl buffer. CL reagents were added to 96-well microtiter plates, which was placed in gas chamber. The gas chamber was sealed and placed in black box of FluorchemTM IS-8800 system, then a certain concentration of NaOH solution was injected into saturated NH4 Cl solution (Fig. 1) and ammonia was produced. The pH of CL reagents in 96-well microtiter plates increased continuously from the same initial value due to the absorbance of ammonia, the relative CL intensity increased with the increasing pH and then CL intensity decreased slowly after reaching maximal intensity. The pH of CL reaction was controlled by the amount of ammonia; the amount of ammonia could be adjusted by varying concentration, pumping time and flow rate of NaOH solution. The light emission kinetics was monitored for at least 30 min with light accumulations lasting 5 min each. A series of image were acquired using Movie Mode of FluorchemTM IS-8800 system. The CL intensity of each well was determined using the Spot Denso function of the software, which combines the pixel intensities. The light emission from each well was quantified by defining a fixed area and counting the number of photon fluxes within this area, the user can manually draw an area of interest through the three OBJECT buttons of software. In this work, the area of each well was enclosed with a box (rectangle). The wells were individually analyzed and the intensities (AVG) were plotted as a function of analytes concentration to yield the calibration curve. IDV is the sum of all the pixel values after background correction: IDV =  (each

pixel value-BACK), AREA is the size (in pixels) of the region enclosed by the box, AVG is the average value after background correction of the pixels enclosed, AVG = IDV/AREA, BACK is the background value that will be subtracted from all the pixels in the object. 2.4.2. Procedure for conventional CL imaging method Conventional CL imaging method was used as contrastive method for reaction-controlled CL imaging. The CL reagents were 50 ␮l luminol solution with p-IP, 50 ␮l H2 O2 solution and 50 ␮l Hb standards, the pH of CL reagents was adjusted to 8.5 by 0.1 mol l−1 Tris–HCl buffer or NaOH solution. The concentration of luminol, p-IP, H2 O2 and Hb standards were same as those in reaction-controlled CL imaging. After addition of the CL reagents (pH 8.5) to 96-well microtiter plates, the plate was placed in black box of the FluorchemTM IS-8800 system and then CL signal was detected and recorded. The exposure time was same as reaction-controlled CL imaging. The background value was obtained by imaging an equally sized region outside the region of interest and was subtracted from each measurement. The intensity of each well was determined using the Spot Denso function of the software. The wells were individually analyzed and the intensities (AVG) were plotted as a function of Hb concentration to yield the calibration curve. The data analysis process was same as reaction-controlled CL imaging. 3. Result and discussion 3.1. Light emission kinetics from luminol-H2 O2 -Hb-p-IP system In order to validate the possibility of reaction-controlled CL imaging, the light emission kinetics from luminol-H2 O2 Hb-p-IP system without pH control by ammonia but by NaOH was collected with a photomultiplier tube of the Type IFFL-D Chemiluminescence Analyzer (Reike, Xi’an). Fig. 2 shows that the CL intensity reached the maximum value

Fig. 2. The light emission kinetics from luminol-H2 O2 -Hb-p-IP (without pH control): Y represents the relative CL intensity; Hb standard, 1.0 × 10−8 mol l−1 ; luminol concentration, 1.25 × 10−3 mol l−1 ; H2 O2 concentration, 3.0 × 10−3 mol l−1 ; p-IP concentration, 1.0 × 10−4 mol l−1 ; high voltage (−700 V); pH, 8.5.

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rapidly and then decreased to a quarter of maximum value within 1.5 min even when p-IP was used. Therefore, the total CL signal cannot be collected without pH control and the maximum CL signal will be missed when 96-wells of microtiter plates were used, which will result in a relative poor sensitivity. 3.2. Optimization of assay buffer and pH for conventional imaging assay As a conjugated protein, hemoglobin consists of four polypeptide subunits and a single heme (iron-porphyrin)

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as the active center. The iron-porphyrin shows similar catalytic function as peroxidase [28]. In this CL imaging system, the assay buffer was medium of enzyme reaction and also was medium of the CL reaction. Different assay buffers such as Tris–HCl, Na2 CO3 –NaHCO3 , NaOH and KH2 PO4 –K2 HPO4 solution were investigated to check their effect on the CL intensity; the result showed that the CL emission in the Tris–HCl buffer was strong and stable than in other buffer solutions. The effect of the pH on the CL intensity was investigated over the range of 7.0–10.0. It was found that CL intensity reached a maximum value when pH was 8.5.

Fig. 3. The CL reaction kinetics of reaction-controlled chemiluminescent image system (typical CCD image of the assay in 96-well plate, the lanes represent three parallel determinations): 1, 2, 3, 4, 5 and 6 represent first, second, third, fourth, fifth and sixth image continuously monitored by a CCD camera; Hb concentration (from a to h: 0, 1.0 × 10–9 , 5.0 × 10−9 , 1.0 × 10−8 , 3.0 × 10−8 , 5.0 × 10−8 , 7.0 × 10−8 and 1.0 × 10−7 mol l−1 ); luminol concentration, 1.25 × 10−3 mol l−1 ; H2 O2 concentration, 3.0 × 10−3 mol l−1 ; p-IP concentration, 1.0 × 10−4 mol l−1 ; exposure time, 5 min; time delay between images, 1 s; pumping time, 10 min; NaOH concentration, 3.0 mol l−1 ; flow rate, 1.0 ml min−1 .

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3.3. Optimization of reaction-controlled detection system 3.3.1. Optimization of image A series of image (Fig. 3) were acquired using Movie Mode of FluorchemTM IS-8800 system, the image with high sensitivity and better reproducibility were selected for analysis, for example, the third image was chosen for analysis in Fig. 3. 3.3.2. Effect of pH, NaOH concentration, pumping time and flow rate on CL intensity In reaction-controlled CL imaging assay, the pH was investigated indirectly. The pH of CL reaction was adjusted by the amount of ammonia; the amount of ammonia was determined by the concentration, pumping time and flow rate of NaOH solution. The influence of NaOH concentration on CL response is illustrated in Fig. 4; the light emission kinetics was monitored for at least 30 min with light accumulations lasting 2 min each. It could be seen that the CL intensity increased upon increasing NaOH concentration until NaOH concentration was 3.0 mol l−1 and then decreased. When NaOH concentration was higher than 3.0 mol l−1 , a lot of ammonia was produced in short time, the pH of CL reaction changed rapidly and the optimum pH could not be kept during exposure time, therefore, the relative CL intensity decreased. Thus, 3.0 mol l−1 NaOH solution was chosen for consequent research work. The effect of pumping time of NaOH solution on the CL intensity was also investigated. When pumping time was too short, little amount of ammonia was produced; CL intensity was low. However, when pumping time was too long, redundant ammonia was produced, the optimum pH could not be kept, thus, the relative CL intensity under certain exposure time decreased. Experimental results showed that maximum CL intensities could be obtained when the pumping time was 10 min. When pumping time was longer than this range, the decrease of CL intensity would occur. The effect of flow rate of NaOH solution on the CL response to Hb was investigated. Flow rates were varied from 1.7 to 0.5 ml min−1 sequentially. Experiment indicated that when the flow rate was 1.0 ml min−1 , system showed the best reproducibility and signal/noise ratio, therefore, 1.0 ml min−1 was chosen for the following study. 3.3.3. Effect of luminol, H2 O2 , p-IP concentration on CL intensity A standard Hb solution of 1.0 × 10−8 mol l−1 was used to optimize the concentration of luminol, H2 O2 and p-IP solution. Initial tests indicated that the concentration of luminol solution had great effect on the detection limit of Hb. Higher luminol concentration brought about a higher baseline, which was deleterious of determining low concentration of Hb. The effect of the concentration of luminol solution on the CL intensity was investigated over the range of 1.25 × 10−4 to 7.5 × 10−3 mol l−1 . It was found that in-

creased CL intensity reached a maximum value when luminol concentration was 1.25 × 10−3 mol l−1 . The effect of H2 O2 concentration on the CL intensity was also investigated. Experimental results showed that when the H2 O2 concentration was 3.0 × 10−3 mol l−1 , the strongest CL signal could be obtained. Under optimum conditions, the CL emission of luminol/Hb/H2 O2 system was enhanced upon addition of pIP solution. Experimental results showed that when p-IP concentration was 1.0 × 10−4 mol l−1 , system showed the best reproducibility and signal/noise ratio. 3.4. Performance of conventional imaging assay without pH control In conventional imaging assay, the concentration of luminol, H2 O2 and p-IP solution was same as those of reactioncontrolled CL imaging assay. NaOH or Tris–HCl solution has been used as medium. When 0.1 mol l−1 Tris–HCl buffer (pH 8.5) was used, CL response to Hb solution was linear in the concentration range of 7.0 × 10−8 to 3.0 × 10−6 mol l−1 and the detection limit was 1.0 × 10−8 mol l−1 (3σ). The regression equation of calibration curve for Hb was ICL = 122.88 [Hb] × 10−8 mol l−1 −81.121, with a correlation coefficient of 0.9975 (n = 6). The relative standard deviation (R.S.D.) for 11 parallel measurements of 7.0 × 10−8 mol l−1 Hb was 6.8%. When NaOH solution (pH 8.5) was used. CL response to Hb solution was linear in the concentration range of 7.0 × 10−8 to 1.0 × 10−6 mol l−1 and the detection limit was 1.0 × 10−8 mol l−1 (3σ). The regression equation of calibration curve for Hb was ICL = 382.51 [Hb] × 10−8 mol l−1 −1061.3, with a correlation coefficient of 0.9953 (n = 5). The relative standard deviation for 11 parallel measurements of 7.0 × 10−8 mol l−1 Hb was 8.2%. Based on above results, it could be seen CL signal with good line range and reproducibilitv could be obtained when Tris–HCl buffer (pH 8.5) was used as medium. 3.5. Performance of the reaction-controlled CL imaging for Hb measurements Under the selected conditions, CL response to Hb solution was linear in the concentration range of 1.0 × 10−9 to 1.0 × 10−7 mol l−1 and the detection limit was 3.0 × 10−10 mol l−1 (3σ). The regression equation of calibration curve for Hb was ICL = 111.34 + 151.88 [Hb] × 10−9 mol l−1 , with a correlation coefficient of 0.9991 (n = 8). The relative standard deviation for 11 parallel measurements of 1.0 × 10−1 mol l−1 Hb was 2.7%. Calibration curve for Hb are shown in Fig. 5, the CL intensities of reaction-controlled CL imaging assay was compared with that of conventional CL imaging assay (the pH was adjusted to 8.5 by Tris–HCl solution in conventional assay); results of Fig. 5 showed that the sensitivity of reaction-controlled CL imaging assay was higher than conventional CL imaging assay, as low as

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The influences of foreign species were also investigated by analyzing a standard solution of 5.0 × 10−9 mol l−1 Hb to which increasing amounts of interfering species were added. The tolerable limit of a foreign species was taken as a relative error not greater than 5%. Studies showed that more than a 1000-fold excess of Na+ , Ca2+ and Cl− ; 500-fold excess of Cu2+ , Co2+ and BSA; and more than 200-fold excess of NO3 − , K+ , H2 PO4 − , CO3 2− and H2 PO4 − did not interfere. 3.6. Sample analysis

Fig. 4. The effect of NaOH concentration on CL intensity (The light emission kinetics was monitored for at least 30 min with light accumulations lasting 2 min each): Hb standard, 1.0 × 10−8 mol l−1 ; flow rate, 1.0 ml min−1 ; pumping time of NaOH solution, 10 min; exposure time for each image, 2 min; luminol concentration, 1.25 × 10−3 mol l−1 ; H2 O2 concentration, 3.0 × 10−3 mol l−1 ; p-IP concentration, 1.0 × 10−4 mol l−1 .

1.0 × 10−9 mol l−1 Hb could be detected by using the proposed model. In contrast, at least 5.0 × 10−8 mol l−1 Hb was required to produce an unambiguous signal in conventional CL imaging method.

The human serum was used directly for the determination after diluting 10 times with water. The accuracy of the Hb was evaluated by determining the recoveries of Hb after adding to fresh human serum. The results obtained by proposed method for the determinations of Hb in serum samples are shown in Table 1.

4. Conclusions A reaction-controlled CL imaging analysis model was established. High sensitivity and better reproducibility were obtained based on this model. It provides a reaction-controlled

Fig. 5. CL intensities comparison between two methods: (1) conventional CL imaging method using 96-well plate: Hb concentration (from a to h: 0, 1.0 × 10−9 , 5.0 × 10−9 , 1.0 × 10−8 , 3.0 × 10−8 , 5.0 × 10−8 , 7.0 × 10−8 and 1.0 × 10−7 mol l−1 ); luminol concentration, 1.25 × 10−3 mol l−1 ; H2 O2 concentration, 3.0 × 10−3 mol l−1 ; p-IP concentration, 1.0 × 10−4 mol l−1 ; exposure time, 5 min; pH, 8.5. (2) Reaction-controlled CL imaging assay using 96-well plate: Hb concentration (from a to h: 0, 1.0 × 10−9 , 5.0 × 10−9 , 1.0 × 10−8 , 3.0 × 10−8 , 5.0 × 10−8 , 7.0 × 10−8 and 1.0 × 10−7 mol l−1 ); luminol concentration, 1.25 × 10−3 mol l−1 ; H2 O2 concentration, 3.0 × 10−3 mol l−1 ; p-IP concentration, 1.0 × 10−4 mol l−1 ; exposure time, 5 min; flow rate, 1.0 ml min−1 ; time delay between images, 1 s; pumping time, 10 min; NaOH concentration, 3.0 mol l−1 . Table 1 Results of recovery test Sample no.

Original (␮g ml−1 )

Added (␮g ml−1 )

Total (␮g ml−1 )

Found (␮g ml−1 )

R.S.D. (%)a

Recovery (%)

1 2 3 4 5

19.0 35.0 25.0 1.9 2.9

3.2 3.2 3.2 1.9 1.9

22.3 38.0 28.0 3.8 4.9

3.3 3.0 3.0 1.9 2.0

3.0 2.2 1.8 2.7 1.9

103 94 94 100 105

a

Average of three determinations.

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model for CL imaging assay and implies the preferable use for immunoassay and clinical chemistry.

Acknowledgement This study was supported by a grant from the Science and Technique Ministry of China (2003BA310A05).

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