ORIGINAL ARTICLE
Comparison between electrochemical ELISA and spectrophotometric ELISA for the detection of dentine sialophosphoprotein for root resorption Hailiang Sha,a Yuxing Bai,b Song Li,c Xiofeng Wang,d and Yajiang Yine Beijing, China
Introduction: Root resorption is an undesirable sequela of orthodontic treatment. It is necessary to establish sensitive methods for identification of teeth at risk for resorption. The x-ray is the traditional method to diagnose root resorption, which is often at a late stage. Some researchers used enzyme-linked immunosorbent immunoassay (ELISA) combined with spectrophotometry to study some biochemical markers of root resorption. However, spectrophotometric detection often has a poor detection limit. Electrochemical detection has inherent advantages over spectrophotometric detection, which is especially suitable for small biologic samples. Methods: We used ELISA combined with electrochemistry and ELISA combined with spectrophotometry to measure the biochemical marker dentine sialophosphoprotein in gingival crevicular fluid of orthodontic patients (treated for 8-12 months). Results: Standard dentine sialophosphoprotein was used to calculate the linear regression equation. No significant difference was found between the electrochemical outcome and the spectrophotometric outcome. But the electrochemical results extended the lower end of detection from 5 pg per milliliter (by spectrophotometry) to 0.5 pg per milliliter. Conclusions: These results showed that ELISA combined with electrochemistry is a reliable and sensitive method to detect dentine sialophosphoprotein in gingival crevicular fluid. (Am J Orthod Dentofacial Orthop 2014;145:36-40)
E
xternal apical root resorption is an overresorption of cement for unknown reasons that can cause loose teeth or even loss of teeth. It is a common sequela of orthodontic treatment and remains unexplained.1,2 According to various studies, about 20% to 100% of patients who receive orthodontic treatment develop root resorption, most of which was mild to moderate, but some was severe. Severe root resorption is a considerable risk factor for the integrity of the a Associate chief physician, Department of Orthodontics, School of Dentistry, Capital Medical University, Beijing, China. b Professor, Department of Orthodontics, School of Dentistry, Capital Medical University, Beijing, China. c Associate professor, Department of Orthodontics, School of Dentistry, Capital Medical University, Beijing, China. d Associate professor, Department of Precision Instruments, Tsinghua University, Beijing, China. e Postgraduate student, Department of Precision Instruments, Tsinghua University, Beijing, China. All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest, and none were reported. Financially supported by the Natural Science Foundation of China (No. 30901700). Address correspondence to: Yuxing Bai, Department of Orthodontics, School of Dentistry, Capital Medical University, 4 Tiantanxili, Dongcheng District, Beijing 100050, P.R. China; e-mail, [email protected]. Submitted, February 2013; revised and accepted, September 2013. 0889-5406/$36.00 Copyright Ó 2014 by the American Association of Orthodontists. http://dx.doi.org/10.1016/j.ajodo.2013.09.008
36
dentition in the long run. Among all teeth, the incisors are most susceptible to resorption because of their root characteristics, which concentrate greater stress on the apexes in orthodontic procedures.3,4 Currently, clinical diagnosis for root resorption is often based on radiographic examination, but it brings radiation exposure, which could limit the longitudinal and systematic study of the roots. Moreover, imaging cannot detect root resorption until 60% to 70% of the mineralized tissue is lost; this makes it impossible to monitor root resorption and stop its progress and can result in loss of teeth.5,6 Therefore, it is necessary to establish sensitive methods to identify at early stages the teeth with a risk of resorption.7 Dentine sialophosphoprotein (DSPP) is a specific protein that is released into the periodontal ligament space during active external root resorption.8 Mah and Prasad9 used biochemical assays to detect dentine proteins and found that dentine phosphoproteins are associated with root resorption. Researchers confirmed that dentine phosphophoryn and dentine sialoprotein in the gingival crevicular fluid (GCF) of people undergoing orthodontic treatment can be regarded as biologic markers for monitoring root resorption.10,11 Dentine sialoprotein and dentine phosphoproteins are
Sha et al
N- and C-terminal proteolytic cleavage products of DSPP, respectively, and belong to the small integrinbinding ligand N-linked glycoprotein family of proteins.6 Hence, DSPP can be regarded as a marker for the detection of root resorption and monitoring its progress.12 Enzyme-linked immunosorbent immunoassay (ELISA) is a traditional method used in biochemical diagnosis and clinical practice. It is easy to use and suitable for most applications.13 But spectrophotometry, the last step to measure the product in ELISA, often has a poor detection limit. In recent years, ELISA combined with electrochemical detection has been proposed and widely used for the detection of tumor markers or plant viruses. It has been shown that electrochemical detection is inherently advantageous over spectrophotometry with its lower detection limit, wider dynamic range, and consequently higher sensitivity.14,15 This was further proved in some studies with small biologic samples.16,17 Obviously, studies with GCF could be limited by its small amount. However, previous studies on DSPP in GCF used ELISA with spectrophotometry, and it was hard to quantify the product.12 We therefore aimed to determine whether electrochemical ELISA is effective in the identification and quantification of DSPP in active orthodontic patients. MATERIAL AND METHODS
Twenty patients were enrolled at the Department of Orthodontics of the dental school affiliated with Capital Medical University in Beijing, China. They were orthodontic patients receiving treatment for 8 to 12 months, including 12 female and 8 male subjects (age range, 1324 years). Inclusion criteria were good oral hygiene, no periodontal disease, no bleeding on probing, no caries, and no systemic diseases. The type of orthodontic displacement of the incisors was not considered in the inclusion criteria because it was not supposed to affect the comparison of the 2 detection methods. This project was approved by the institutional review board of Beijing Stomatological Hospital, and informed consents were obtained from the patients. For each patient, 2 teeth were chosen for GCF collection: the left and right maxillary central incisors. The teeth were gently washed with water, dried, and isolated with cotton rolls to prevent saliva contamination. GCF was collected from the mesial and distal sides of each tooth with a filter paper strip (Tianjin Zhongjin Biological Technology, Tianjin, China), which was inserted 1 to 2 mm into the gingival sulcus for 30 seconds. The same procedure was repeated at a 1-minute interval. As a result, 160 strips of samples were obtained, 2 for each position. After removal, each strip
37
was immediately sealed in a microcentrifuge tube with phosphate-buffered saline solution containing 0.1 m mol/L of phenylmethylsulphonyl fluoride. To retrieve the sample from the paper strip, the GCF was eluted by centrifugal filtration at 15,000 g for 5 minutes with 100 mL aliquots of buffer. Two GCF samples from the same position were then pooled to give a total volume of 200 mL and stored at 70 C.18 Therefore, 80 GCF samples were prepared, and each was assessed with both electrochemical and spectrophotometric ELISA. The sandwich ELISA procedure was used for the detection of DSPP in the standard solution of all GCF samples. In general, 50 mL of the DSPP sample was mixed with biotin labeled DSPP antibody and added into the microplate, which was precoated with DSPP monoclonal antibody. Then 50 mL of streptavidin labeled horseradish peroxidase solution was added into each well. After incubating at 37 C for 60 minutes, the wells of the microplate were washed 5 times with 0.02 mol/L of phosphate-buffered saline solution containing 0.05% Tween-20. After washing, 200 mL of substrate solution was added into each well and incubated at 37 C for 30 minutes. Then 50 mL of Britton-Robinson buffer (0.2 mol/L, pH 5.0) was added to each well. For the electrochemical analysis of the samples, a miniature 3-electrode system was directly inserted into the well, and the second-order derivative linear sweep voltammetric reduction peak was measured with a JP303 voltammetric analyzer (Chengdu Apparatus, Chengdu, China). The experimental procedure is shown in Figure 1. Using horseradish peroxidase as the labeled enzyme, o-tilidine was used as the enzymatic reaction substrate in this research; it can be oxidized by hydrogen peroxide to give an electroactive azo product. Under the optimal conditions, the proposed o-tilidine–hydrogen peroxide–horseradish peroxidase system was combined with double-antibody sandwich ELISA and further used to detect DSPP in GCF. In the electrochemical ELISA procedure described above, the more DSPP in the solution of GCF, the more the labeled horseradish peroxidase was coupled on the microplate and more of the enzymatic product could be formed; this gave a higher reduction peak current. The prepared GCF solution of each sample was also analyzed with the traditional spectrophotometric ELISA procedure. In the spectrophotometric ELISA procedure, the more DSPP in the solution of GCF, the more the labeled horseradish peroxidase was coupled on the microplate and more of the enzymatic product could be formed; this gave a darker color.
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Fig 1. Procedures for the sandwich ELISA for DSPP.
Statistical analysis
The linear regression equation of DSPP was calculated with standard DSPP solution (Shanghai Jingtian Biological Technology, Shanghai, China), with reference to the range of DSPP shown in previous studies, to further establish the electrochemical method for the detection of concentrations of DSPP. Then the DSPP concentrations in the GCF samples measured by the electrochemical ELISA procedure were compared with those by the spectroscopic ELISA procedure. The analysis was performed with a paired-samples rank sum test, and the level of significance was set at P 5 0.05. RESULTS
The second-order derivative linear sweep voltammetric peak current was linear with DSPP concentration in the range of 0.5 to 800.0 pg per milliliter with the linear regression equation: Ip}(mA) 5 0.0974 1 2.030 3 10 4 C(pg/mL) (n 5 7, g 5 0.9991) (Fig 2). We found that the detection limit of the electrochemical method (0.5 pg/mL [3s]) was 10 times lower than that of the spectroscopic method (5.0 pg/mL [3s]), and the range was 5.0 to 1500.0 pg per milliliter (data from ELISA kit of Shanghai Jingtian Biological Technology). This indicated a higher sensitivity of the electrochemical method. The paired-samples rank sum test showed no significant difference between the DSPP concentrations in the GCF samples measured by the spectroscopic ELISA procedure and those measured by the electrochemical ELISA
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procedure (P 5 0.500) (Table I). The DSPP concentrations of the patients detected by electrochemical ELISA (median, 485.90 pg/mL; range, 314.2-671.3 pg/mL) were close to those detected by the traditional spectrophotometric ELISA (median, 490.20 pg/mL; range, 301.4-668.2 pg/mL). DISCUSSION
This study shows that electrochemical ELISA is as accurate as spectrophotometric ELISA in detecting DSPP in the GCF of patients receiving orthodontic treatment. There was no significant difference between the DSPP concentrations by the electrochemical method and the traditional spectroscopic method. The reliability was also evidenced by the similarity of the DSPP concentrations between the electrochemical results (median, 485.90 pg/mL) and the spectroscopic results (median, 490.20 pg/mL). In term of sensitivity, electrochemical ELISA has the potential to be more sensitive in detecting DSPP because it extended the low end of the detection range from 5.0 pg per milliliter by spectroscopy to 0.5 pg per milliliter. In the spectrophotometric analysis, the concentration of product is related to the intensity of the transmitted light on the spectrophotometer. However, the logarithmic relationship of optical density with transmittance and a deficient light-source density on the spectrophotometer could limit the accuracy and sensitivity of traditional spectrophotometric ELISA.15
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Table I. Comparison of electrochemical with spectroscopic results for DSPP in GCF samples SELISA median (pg/mL) 490.20
EELISA median (pg/mL) 485.90
SELISAEELISA Z 0.674
SELISAEELISA 2-tailed P 0.500
SELISA, Spectrophotometric ELISA; EELISA, electrochemical ELISA.
Fig 2. Calibration plot for detection of DSPP by electrochemical ELISA.
Electrochemical ELISA is a new application in orthodontics; it integrates the specificity of immunoassays with the sensitivity of electrochemical analysis.16 The concentration of product is related to the peak current of the electrochemical analyzer, which is not influenced by the light density of the analyzer or the turbidity of the samples. Therefore, electrochemical detection is more suitable to measure small biologic samples. In the electrochemical procedure of this study, it was critical to choose the right enzymatic reaction substrate and optimal conditions. As a substance that can be easily oxidized by hydrogen peroxide with the catalysis of horseradish peroxidase, o-tilidine is a suitable substrate for the horseradish peroxidase reaction system with the electroactive product that resulted. The optimal concentrations of hydrogen peroxide and o-tilidine and the effect of pH of the supporting electrolyte on the electrochemical detection for the enzymatic product were investigated to ascertain optimal conditions. Under the selected conditions, the second-order derivative linear sweep voltammetric peak current was linear with DSPP concentration in the range of 0.5 to 800.0 pg per milliliter. The detection limit was calculated as 0.5 pg per milliliter, which was 10 times lower than the traditional spectrophotometric method. This indicated the higher sensitivity of the electrochemical method; hence, it can be used for samples with lower concentrations. As for the cost of electrochemical ELISA, except for the needed reagents, the major apparatus of electrochemical ELISA is a voltammetric analyzer. The price of the analyzer is similar to that of the commonly used UV-Vis spectrophotometer. So, compared with the cost of spectrophotometric measurement, the cost of electrochemical detection is reasonable.
For root resorption that is a common sequela of orthodontic treatment, the diagnosis helps orthodontists to modify their treatment plans to delay, stop, or reverse the resorption.1,2 They often resort to x-ray imaging for its identification; unfortunately, this can show the resorption only when it becomes severe (60%-70%). DSPP, including dentine sialoprotein and dentine phosphoproteins, as the specific proteins of root resorption, has been demonstrated as an effective marker for resorption by previous researchers.8,10 In their reports, ELISA was combined with spectroscopy to measure the concentrations of dentine sialoprotein and dentine phosphoproteins. To our best knowledge, this study is the first to use electrochemistry instead of spectroscopy to quantify the concentration of DSPP, which was proved to be reliable with high sensitivity. This study suggests that electrochemical ELISA could be a new way to detect DSPP in GCF to diagnose root resorption in time. It is noninvasive and facilitates continuous and systematic monitoring of the resorption progression. Early diagnosis of root resorption is vital for its correction and consequently any change in the orthodontic treatment plan. Electrochemical ELISA detection has inherent advantages over spectrophotometric ELISA, with its lower detection limit, wider dynamic range, and consequently higher sensitivity.13,15 The amount of GCF is small; this could limit studies on GCF. A more sensitive method of detection, such as electrochemistry, is much superior to spectroscopy in the investigation of GCF. In our study, the absence of a significant difference between the electrochemical results and the spectroscopic results suggests the reliability of the electrochemical method. However, it could suggest that the choice of GCF samples was limited, with patients treated for less than 8 months excluded. If these patients, who might be undergoing preliminary root resorption, were included, a significant difference might have been produced. We speculate that if a wider range of subjects, especially patients treated for less than 8 months, were included, this could show the progression of root resorption. This could be enabled with the more sensitive electrochemical method. Also, the sensitivity of electrochemical ELISA could enable some studies in
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orthodontics, cross-sectional or longitudinal, concerning the variations of low concentrations. CONCLUSIONS
Electrochemical ELISA is an applicable, reliable, and more sensitive method for the detection of the biochemical marker DSPP for root resorption. It could help facilitate a continuous longitudinal study to determine the etiology of root resorption. This new method might help future studies to look at orthodontic patients who received shorter treatment (\8 months) and might have preliminary root resorption. Its application might also be extended to some small, low-concentration biologic samples. REFERENCES 1. Brezniak N, Wasserstein A. Root resorption after orthodontic treatment: part 1. Literature review. Am J Orthod Dentofacial Orthop 1993;103:62-6. 2. Brezniak N, Wasserstein A. Orthodontically induced inflammatory root resorption. Part II: the clinical aspects. Angle Orthod 2002;72: 180-4. 3. Rudolph DJ, Willes PMG, Sameshima GT. A finite element model of apical force distribution from orthodontic tooth movement. Angle Orthod 2001;71:127-31. 4. Shaw AM, Sameshima GT, Vu HV. Mechanical stress generated by orthodontic forces on apical root cementum: a finite element model. Orthod Craniofac Res 2004;7:98-107. 5. Andreasen FM, Sewerin I, Mandel U, Andreasen JO. Radiographic assessment of simulated root resorption cavities. Endod Dent Traumatol 1987;3:21-7. 6. Chapnick L. External root resorption: an experimental radiographic evaluation. Oral Surg Oral Med Oral Pathol 1989;67:578-82.
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7. Levander E, Malmgren O. Evaluation of the risk of root resorption during orthodontic treatment: a study of upper incisors. Eur J Orthod 1988;10:30-8. 8. Ritchie HH, Wang LH. Sequence determination of an extremely acidic rat dentine phosphoprotein. J Biol Chem 1996;271:21695-8. 9. Mah J, Prasad N. Dentine phosphoproteins in gingival crevicular fluid during root resorption. Eur J Orthod 2004;26:25-30. 10. Balducci L, Ramachandran A, Hao J, Narayanan K, Evans C, George A. Biological markers for evaluation of root resorption. Arch Oral Biol 2007;52:203-8. 11. Kereshanan S, Stephenson P, Waddington R. Identification of dentine sialoprotein in gingival crevicular fluid during physiological root resorption and orthodontic tooth movement. Eur J Orthod 2008;30:307-14. 12. Zainal Ariffin SH, Yamamoto Z, Zainol Abidin IZ, Megat Abdul Wahab R, Zainal Ariffin Z. Cellular and molecular changes in orthodontic tooth movement. ScientificWorldJournal 2011; 11:1788-803. 13. Tijssen P. Practice and theory of enzyme immunoassays. Amsterdam, The Netherlands: Elsevier Science Publishers B.V. (Biomedical Division); 1985. p. 95-108. 14. Jiao K, Sun W. Application of p-phenylenediamine as an electrochemical substrate in peroxidase-mediated voltammetric enzyme immunoassay. Anal Chim Acta 2000;413:71-8. 15. Zhang SS, Yang J, Lin JH. 3, 3'-diaminobenzidine (DAB)-H2O2HRP voltammetric enzyme-linked immunoassay for the detection of carcionembryonic antigen. Bioelectrochemistry 2008;72:47-52. 16. Draisci R, delli Quadri F, Achene L, Volpe G, Palleschi L, Palleschi G. A new electrochemical enzyme-linked immunosorbent assay for the screening of macrolide antibiotic residues in bovine meat. Analyst 2001;126:1942-6. 17. Jiao K, Sun W, Zhang SS. Sensitive detection of a plant virus by electrochemical enzyme-linked immunoassay. Fresenius J Anal Chem 2000;367:667-71. 18. Offenbacher S, Odle BM, Van Dyke TE. The use of crevicular fluid prostaglandin E2 levels as a predictor of periodontal attachment loss. J Periodont Res 1986;21:101-12.
American Journal of Orthodontics and Dentofacial Orthopedics
Comparison between electrochemical ELISA and spectrophotometric ELISA for the detection of dentine sialophosphoprotein for root resorption Hailiang Sha,a Yuxing Bai,b Song Li,c Xiofeng Wang,d and Yajiang Yine Beijing, China
Introduction: Root resorption is an undesirable sequela of orthodontic treatment. It is necessary to establish sensitive methods for identification of teeth at risk for resorption. The x-ray is the traditional method to diagnose root resorption, which is often at a late stage. Some researchers used enzyme-linked immunosorbent immunoassay (ELISA) combined with spectrophotometry to study some biochemical markers of root resorption. However, spectrophotometric detection often has a poor detection limit. Electrochemical detection has inherent advantages over spectrophotometric detection, which is especially suitable for small biologic samples. Methods: We used ELISA combined with electrochemistry and ELISA combined with spectrophotometry to measure the biochemical marker dentine sialophosphoprotein in gingival crevicular fluid of orthodontic patients (treated for 8-12 months). Results: Standard dentine sialophosphoprotein was used to calculate the linear regression equation. No significant difference was found between the electrochemical outcome and the spectrophotometric outcome. But the electrochemical results extended the lower end of detection from 5 pg per milliliter (by spectrophotometry) to 0.5 pg per milliliter. Conclusions: These results showed that ELISA combined with electrochemistry is a reliable and sensitive method to detect dentine sialophosphoprotein in gingival crevicular fluid. (Am J Orthod Dentofacial Orthop 2014;145:36-40)
E
xternal apical root resorption is an overresorption of cement for unknown reasons that can cause loose teeth or even loss of teeth. It is a common sequela of orthodontic treatment and remains unexplained.1,2 According to various studies, about 20% to 100% of patients who receive orthodontic treatment develop root resorption, most of which was mild to moderate, but some was severe. Severe root resorption is a considerable risk factor for the integrity of the a Associate chief physician, Department of Orthodontics, School of Dentistry, Capital Medical University, Beijing, China. b Professor, Department of Orthodontics, School of Dentistry, Capital Medical University, Beijing, China. c Associate professor, Department of Orthodontics, School of Dentistry, Capital Medical University, Beijing, China. d Associate professor, Department of Precision Instruments, Tsinghua University, Beijing, China. e Postgraduate student, Department of Precision Instruments, Tsinghua University, Beijing, China. All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest, and none were reported. Financially supported by the Natural Science Foundation of China (No. 30901700). Address correspondence to: Yuxing Bai, Department of Orthodontics, School of Dentistry, Capital Medical University, 4 Tiantanxili, Dongcheng District, Beijing 100050, P.R. China; e-mail, [email protected]. Submitted, February 2013; revised and accepted, September 2013. 0889-5406/$36.00 Copyright Ó 2014 by the American Association of Orthodontists. http://dx.doi.org/10.1016/j.ajodo.2013.09.008
36
dentition in the long run. Among all teeth, the incisors are most susceptible to resorption because of their root characteristics, which concentrate greater stress on the apexes in orthodontic procedures.3,4 Currently, clinical diagnosis for root resorption is often based on radiographic examination, but it brings radiation exposure, which could limit the longitudinal and systematic study of the roots. Moreover, imaging cannot detect root resorption until 60% to 70% of the mineralized tissue is lost; this makes it impossible to monitor root resorption and stop its progress and can result in loss of teeth.5,6 Therefore, it is necessary to establish sensitive methods to identify at early stages the teeth with a risk of resorption.7 Dentine sialophosphoprotein (DSPP) is a specific protein that is released into the periodontal ligament space during active external root resorption.8 Mah and Prasad9 used biochemical assays to detect dentine proteins and found that dentine phosphoproteins are associated with root resorption. Researchers confirmed that dentine phosphophoryn and dentine sialoprotein in the gingival crevicular fluid (GCF) of people undergoing orthodontic treatment can be regarded as biologic markers for monitoring root resorption.10,11 Dentine sialoprotein and dentine phosphoproteins are
Sha et al
N- and C-terminal proteolytic cleavage products of DSPP, respectively, and belong to the small integrinbinding ligand N-linked glycoprotein family of proteins.6 Hence, DSPP can be regarded as a marker for the detection of root resorption and monitoring its progress.12 Enzyme-linked immunosorbent immunoassay (ELISA) is a traditional method used in biochemical diagnosis and clinical practice. It is easy to use and suitable for most applications.13 But spectrophotometry, the last step to measure the product in ELISA, often has a poor detection limit. In recent years, ELISA combined with electrochemical detection has been proposed and widely used for the detection of tumor markers or plant viruses. It has been shown that electrochemical detection is inherently advantageous over spectrophotometry with its lower detection limit, wider dynamic range, and consequently higher sensitivity.14,15 This was further proved in some studies with small biologic samples.16,17 Obviously, studies with GCF could be limited by its small amount. However, previous studies on DSPP in GCF used ELISA with spectrophotometry, and it was hard to quantify the product.12 We therefore aimed to determine whether electrochemical ELISA is effective in the identification and quantification of DSPP in active orthodontic patients. MATERIAL AND METHODS
Twenty patients were enrolled at the Department of Orthodontics of the dental school affiliated with Capital Medical University in Beijing, China. They were orthodontic patients receiving treatment for 8 to 12 months, including 12 female and 8 male subjects (age range, 1324 years). Inclusion criteria were good oral hygiene, no periodontal disease, no bleeding on probing, no caries, and no systemic diseases. The type of orthodontic displacement of the incisors was not considered in the inclusion criteria because it was not supposed to affect the comparison of the 2 detection methods. This project was approved by the institutional review board of Beijing Stomatological Hospital, and informed consents were obtained from the patients. For each patient, 2 teeth were chosen for GCF collection: the left and right maxillary central incisors. The teeth were gently washed with water, dried, and isolated with cotton rolls to prevent saliva contamination. GCF was collected from the mesial and distal sides of each tooth with a filter paper strip (Tianjin Zhongjin Biological Technology, Tianjin, China), which was inserted 1 to 2 mm into the gingival sulcus for 30 seconds. The same procedure was repeated at a 1-minute interval. As a result, 160 strips of samples were obtained, 2 for each position. After removal, each strip
37
was immediately sealed in a microcentrifuge tube with phosphate-buffered saline solution containing 0.1 m mol/L of phenylmethylsulphonyl fluoride. To retrieve the sample from the paper strip, the GCF was eluted by centrifugal filtration at 15,000 g for 5 minutes with 100 mL aliquots of buffer. Two GCF samples from the same position were then pooled to give a total volume of 200 mL and stored at 70 C.18 Therefore, 80 GCF samples were prepared, and each was assessed with both electrochemical and spectrophotometric ELISA. The sandwich ELISA procedure was used for the detection of DSPP in the standard solution of all GCF samples. In general, 50 mL of the DSPP sample was mixed with biotin labeled DSPP antibody and added into the microplate, which was precoated with DSPP monoclonal antibody. Then 50 mL of streptavidin labeled horseradish peroxidase solution was added into each well. After incubating at 37 C for 60 minutes, the wells of the microplate were washed 5 times with 0.02 mol/L of phosphate-buffered saline solution containing 0.05% Tween-20. After washing, 200 mL of substrate solution was added into each well and incubated at 37 C for 30 minutes. Then 50 mL of Britton-Robinson buffer (0.2 mol/L, pH 5.0) was added to each well. For the electrochemical analysis of the samples, a miniature 3-electrode system was directly inserted into the well, and the second-order derivative linear sweep voltammetric reduction peak was measured with a JP303 voltammetric analyzer (Chengdu Apparatus, Chengdu, China). The experimental procedure is shown in Figure 1. Using horseradish peroxidase as the labeled enzyme, o-tilidine was used as the enzymatic reaction substrate in this research; it can be oxidized by hydrogen peroxide to give an electroactive azo product. Under the optimal conditions, the proposed o-tilidine–hydrogen peroxide–horseradish peroxidase system was combined with double-antibody sandwich ELISA and further used to detect DSPP in GCF. In the electrochemical ELISA procedure described above, the more DSPP in the solution of GCF, the more the labeled horseradish peroxidase was coupled on the microplate and more of the enzymatic product could be formed; this gave a higher reduction peak current. The prepared GCF solution of each sample was also analyzed with the traditional spectrophotometric ELISA procedure. In the spectrophotometric ELISA procedure, the more DSPP in the solution of GCF, the more the labeled horseradish peroxidase was coupled on the microplate and more of the enzymatic product could be formed; this gave a darker color.
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Fig 1. Procedures for the sandwich ELISA for DSPP.
Statistical analysis
The linear regression equation of DSPP was calculated with standard DSPP solution (Shanghai Jingtian Biological Technology, Shanghai, China), with reference to the range of DSPP shown in previous studies, to further establish the electrochemical method for the detection of concentrations of DSPP. Then the DSPP concentrations in the GCF samples measured by the electrochemical ELISA procedure were compared with those by the spectroscopic ELISA procedure. The analysis was performed with a paired-samples rank sum test, and the level of significance was set at P 5 0.05. RESULTS
The second-order derivative linear sweep voltammetric peak current was linear with DSPP concentration in the range of 0.5 to 800.0 pg per milliliter with the linear regression equation: Ip}(mA) 5 0.0974 1 2.030 3 10 4 C(pg/mL) (n 5 7, g 5 0.9991) (Fig 2). We found that the detection limit of the electrochemical method (0.5 pg/mL [3s]) was 10 times lower than that of the spectroscopic method (5.0 pg/mL [3s]), and the range was 5.0 to 1500.0 pg per milliliter (data from ELISA kit of Shanghai Jingtian Biological Technology). This indicated a higher sensitivity of the electrochemical method. The paired-samples rank sum test showed no significant difference between the DSPP concentrations in the GCF samples measured by the spectroscopic ELISA procedure and those measured by the electrochemical ELISA
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procedure (P 5 0.500) (Table I). The DSPP concentrations of the patients detected by electrochemical ELISA (median, 485.90 pg/mL; range, 314.2-671.3 pg/mL) were close to those detected by the traditional spectrophotometric ELISA (median, 490.20 pg/mL; range, 301.4-668.2 pg/mL). DISCUSSION
This study shows that electrochemical ELISA is as accurate as spectrophotometric ELISA in detecting DSPP in the GCF of patients receiving orthodontic treatment. There was no significant difference between the DSPP concentrations by the electrochemical method and the traditional spectroscopic method. The reliability was also evidenced by the similarity of the DSPP concentrations between the electrochemical results (median, 485.90 pg/mL) and the spectroscopic results (median, 490.20 pg/mL). In term of sensitivity, electrochemical ELISA has the potential to be more sensitive in detecting DSPP because it extended the low end of the detection range from 5.0 pg per milliliter by spectroscopy to 0.5 pg per milliliter. In the spectrophotometric analysis, the concentration of product is related to the intensity of the transmitted light on the spectrophotometer. However, the logarithmic relationship of optical density with transmittance and a deficient light-source density on the spectrophotometer could limit the accuracy and sensitivity of traditional spectrophotometric ELISA.15
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Table I. Comparison of electrochemical with spectroscopic results for DSPP in GCF samples SELISA median (pg/mL) 490.20
EELISA median (pg/mL) 485.90
SELISAEELISA Z 0.674
SELISAEELISA 2-tailed P 0.500
SELISA, Spectrophotometric ELISA; EELISA, electrochemical ELISA.
Fig 2. Calibration plot for detection of DSPP by electrochemical ELISA.
Electrochemical ELISA is a new application in orthodontics; it integrates the specificity of immunoassays with the sensitivity of electrochemical analysis.16 The concentration of product is related to the peak current of the electrochemical analyzer, which is not influenced by the light density of the analyzer or the turbidity of the samples. Therefore, electrochemical detection is more suitable to measure small biologic samples. In the electrochemical procedure of this study, it was critical to choose the right enzymatic reaction substrate and optimal conditions. As a substance that can be easily oxidized by hydrogen peroxide with the catalysis of horseradish peroxidase, o-tilidine is a suitable substrate for the horseradish peroxidase reaction system with the electroactive product that resulted. The optimal concentrations of hydrogen peroxide and o-tilidine and the effect of pH of the supporting electrolyte on the electrochemical detection for the enzymatic product were investigated to ascertain optimal conditions. Under the selected conditions, the second-order derivative linear sweep voltammetric peak current was linear with DSPP concentration in the range of 0.5 to 800.0 pg per milliliter. The detection limit was calculated as 0.5 pg per milliliter, which was 10 times lower than the traditional spectrophotometric method. This indicated the higher sensitivity of the electrochemical method; hence, it can be used for samples with lower concentrations. As for the cost of electrochemical ELISA, except for the needed reagents, the major apparatus of electrochemical ELISA is a voltammetric analyzer. The price of the analyzer is similar to that of the commonly used UV-Vis spectrophotometer. So, compared with the cost of spectrophotometric measurement, the cost of electrochemical detection is reasonable.
For root resorption that is a common sequela of orthodontic treatment, the diagnosis helps orthodontists to modify their treatment plans to delay, stop, or reverse the resorption.1,2 They often resort to x-ray imaging for its identification; unfortunately, this can show the resorption only when it becomes severe (60%-70%). DSPP, including dentine sialoprotein and dentine phosphoproteins, as the specific proteins of root resorption, has been demonstrated as an effective marker for resorption by previous researchers.8,10 In their reports, ELISA was combined with spectroscopy to measure the concentrations of dentine sialoprotein and dentine phosphoproteins. To our best knowledge, this study is the first to use electrochemistry instead of spectroscopy to quantify the concentration of DSPP, which was proved to be reliable with high sensitivity. This study suggests that electrochemical ELISA could be a new way to detect DSPP in GCF to diagnose root resorption in time. It is noninvasive and facilitates continuous and systematic monitoring of the resorption progression. Early diagnosis of root resorption is vital for its correction and consequently any change in the orthodontic treatment plan. Electrochemical ELISA detection has inherent advantages over spectrophotometric ELISA, with its lower detection limit, wider dynamic range, and consequently higher sensitivity.13,15 The amount of GCF is small; this could limit studies on GCF. A more sensitive method of detection, such as electrochemistry, is much superior to spectroscopy in the investigation of GCF. In our study, the absence of a significant difference between the electrochemical results and the spectroscopic results suggests the reliability of the electrochemical method. However, it could suggest that the choice of GCF samples was limited, with patients treated for less than 8 months excluded. If these patients, who might be undergoing preliminary root resorption, were included, a significant difference might have been produced. We speculate that if a wider range of subjects, especially patients treated for less than 8 months, were included, this could show the progression of root resorption. This could be enabled with the more sensitive electrochemical method. Also, the sensitivity of electrochemical ELISA could enable some studies in
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orthodontics, cross-sectional or longitudinal, concerning the variations of low concentrations. CONCLUSIONS
Electrochemical ELISA is an applicable, reliable, and more sensitive method for the detection of the biochemical marker DSPP for root resorption. It could help facilitate a continuous longitudinal study to determine the etiology of root resorption. This new method might help future studies to look at orthodontic patients who received shorter treatment (\8 months) and might have preliminary root resorption. Its application might also be extended to some small, low-concentration biologic samples. REFERENCES 1. Brezniak N, Wasserstein A. Root resorption after orthodontic treatment: part 1. Literature review. Am J Orthod Dentofacial Orthop 1993;103:62-6. 2. Brezniak N, Wasserstein A. Orthodontically induced inflammatory root resorption. Part II: the clinical aspects. Angle Orthod 2002;72: 180-4. 3. Rudolph DJ, Willes PMG, Sameshima GT. A finite element model of apical force distribution from orthodontic tooth movement. Angle Orthod 2001;71:127-31. 4. Shaw AM, Sameshima GT, Vu HV. Mechanical stress generated by orthodontic forces on apical root cementum: a finite element model. Orthod Craniofac Res 2004;7:98-107. 5. Andreasen FM, Sewerin I, Mandel U, Andreasen JO. Radiographic assessment of simulated root resorption cavities. Endod Dent Traumatol 1987;3:21-7. 6. Chapnick L. External root resorption: an experimental radiographic evaluation. Oral Surg Oral Med Oral Pathol 1989;67:578-82.
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7. Levander E, Malmgren O. Evaluation of the risk of root resorption during orthodontic treatment: a study of upper incisors. Eur J Orthod 1988;10:30-8. 8. Ritchie HH, Wang LH. Sequence determination of an extremely acidic rat dentine phosphoprotein. J Biol Chem 1996;271:21695-8. 9. Mah J, Prasad N. Dentine phosphoproteins in gingival crevicular fluid during root resorption. Eur J Orthod 2004;26:25-30. 10. Balducci L, Ramachandran A, Hao J, Narayanan K, Evans C, George A. Biological markers for evaluation of root resorption. Arch Oral Biol 2007;52:203-8. 11. Kereshanan S, Stephenson P, Waddington R. Identification of dentine sialoprotein in gingival crevicular fluid during physiological root resorption and orthodontic tooth movement. Eur J Orthod 2008;30:307-14. 12. Zainal Ariffin SH, Yamamoto Z, Zainol Abidin IZ, Megat Abdul Wahab R, Zainal Ariffin Z. Cellular and molecular changes in orthodontic tooth movement. ScientificWorldJournal 2011; 11:1788-803. 13. Tijssen P. Practice and theory of enzyme immunoassays. Amsterdam, The Netherlands: Elsevier Science Publishers B.V. (Biomedical Division); 1985. p. 95-108. 14. Jiao K, Sun W. Application of p-phenylenediamine as an electrochemical substrate in peroxidase-mediated voltammetric enzyme immunoassay. Anal Chim Acta 2000;413:71-8. 15. Zhang SS, Yang J, Lin JH. 3, 3'-diaminobenzidine (DAB)-H2O2HRP voltammetric enzyme-linked immunoassay for the detection of carcionembryonic antigen. Bioelectrochemistry 2008;72:47-52. 16. Draisci R, delli Quadri F, Achene L, Volpe G, Palleschi L, Palleschi G. A new electrochemical enzyme-linked immunosorbent assay for the screening of macrolide antibiotic residues in bovine meat. Analyst 2001;126:1942-6. 17. Jiao K, Sun W, Zhang SS. Sensitive detection of a plant virus by electrochemical enzyme-linked immunoassay. Fresenius J Anal Chem 2000;367:667-71. 18. Offenbacher S, Odle BM, Van Dyke TE. The use of crevicular fluid prostaglandin E2 levels as a predictor of periodontal attachment loss. J Periodont Res 1986;21:101-12.
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