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Respective and simultaneous detection tumor markers CA125 and STIP1 using aptamer-based fluorescent and RLS sensors
Accepted Manuscript Title: Respective and simultaneous detection tumor markers CA125 and STIP1 using aptamer-based fluorescent and RLS sensors Authors: Feng Chen, Yi Liu, Chunyan Chen, Hang Gong, Changqun Cai, Xiaoming Chen PII: DOI: Reference:
S0925-4005(17)30162-4 http://dx.doi.org/doi:10.1016/j.snb.2017.01.155 SNB 21680
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
6-12-2016 16-1-2017 24-1-2017
Please cite this article as: Feng Chen, Yi Liu, Chunyan Chen, Hang Gong, Changqun Cai, Xiaoming Chen, Respective and simultaneous detection tumor markers CA125 and STIP1 using aptamer-based fluorescent and RLS sensors, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2017.01.155 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Respective and simultaneous detection tumor markers CA125 and STIP1 using aptamer-based fluorescent and RLS sensors Feng Chen, Yi Liu, Chunyan Chen, Hang Gong*, Changqun Cai*, Xiaoming Chen Key Laboratory of Environmentally Friendly Chemistry and Applications of Ministry of Education, College of Chemistry, Xiangtan University, Xiangtan, Hunan 411105, China. Corresponding author. Tel: +86 152 7321 9560. Fax: +86-732-5829-2251. Email: [email protected] (H.Gong); [email protected] (C.Cai).
Graphical abstract:
Tumor marker CA125 and STIP1 were respectively and simultaneously detected using aptamers with fluorescence based detection on RGO as well as RLS detection for screening ovarian cancer.
Highlighs:
A novel strategy for screening ovarian cancer is proposed.
The strategy is individual and simultaneous detection of tumor marker CA125 and STIP1 using fluorescence intensity and resonance light scattering.
The detection for CA125 and STIP1 is using aptamer as binding element for the first time.
Abstract Here we reported a method for the early diagnosis of ovarian cancer that is respective and simultaneous detection of tumour marker cancer antigen 125 (CA125) and stress-induced phosphoprotein 1 (STIP1) using aptamer-based fluorescent and RLS sensors. The fluorescent sensor was fabricated by Carboxyfluorescein (FAM)-labelled CA125 aptamer and cyanine-5-modified STIP1 aptamer onto the surface of RGO, thereby quenching the fluorescence of fluorophores. When STIP1 was introduced, the fluorescence recovered. When CA125 was added, the specific binding of aptamer and CA125 triggered the release of the FAM-labeled CA125 aptamer under the effect of formaldehyde, and adsorbed on the surface of RGO which lead to fluorescence quenching of FAM. This fluorescence signals change could be used to detect CA125 and STIP1 with the lowest detectable concentration down to 0.05 U⋅mL-1 and 1 ng⋅mL-1 respectively. The RLS sensor was fabricated by methyl violet interact with dsDNA which is obtained by CA125 aptamer hybridized to STIP1 aptamer. The dependence of RLS intensity on targets amount was successfully utilized for simultaneous detection of CA125 and STIP1, which provide robust evidences for the presence of ovarian cancer in the early stage. This strategy is reliable, sensitive, and may form the basis for rapid screening of ovarian cancer. Keywords: Aptamer; Resonance light scattering sensor; Early diagnosis of ovarian cancer; Cancer antigen 125; Stress-induced phosphoprotein 1
1. Introduction Ovarian cancer is the leading cause of gynaecological cancer death [1]. The high case fatality rate is mainly attributed to its nonspecific symptoms and lack of an adequate physical exam; over 75 percent of cases are diagnosed in the late stages when the disease has metastasized beyond the primary site, leading to a five year survival rate of less than 30 percent [2]. Since the first characterization of a monoclonal antibody against a glycoprotein, CA125, in 1981 [3], measurement of serum levels of CA125 has become standard practice for the preoperative evaluation of ovarian masses [4]. However, the sensitivity of CA125 is only about 80% for detecting overall ovarian cancer [5] and only about 50% for early stage cancer [6]. In clinical trials, most abnormal CA125 values are false positives that may be caused by benign ovarian disease [7] or benign fluid effusion [8] or hepatopathies with ascites [9] or Meigs’syndrome [10]. These findings suggest that CA125 alone is not an appropriate surrogate marker for the diagnosis and screening of ovarian cancer. Wang et al. reported that STIP1 was secreted by ovarian cancer tissues into the peripheral blood of patients and the combined measurement of CA125 and STIP1 has been shown to improve the early detection of ovarian cancer [11]. The combined use of CA125﹥35 U⋅mL-1 and STIP1﹥55 ng⋅mL-1 in detecting ovarian cancer, resulted in 100% sensitivity, 95.3% specificity, 95.6% positive predictive value, and 100% negative predictive value. Hence, it is desired to achieve simultaneous detection of CA125 and STIP1 which provide robust evidences for the presence of ovarian cancer in the early stage.
CA125 plays vital role in the diagnosis of ovarian cancer. For clinical laboratory, CA125 levels are usually measured by radiometric[12] and enzyme immunoassay[13]. These conventional assays are time-consuming and required special equipped laboratories. In recent decades, several assays have been developed to detect CA125, including surface plasmon resonance[14], electrochemistry[15, 16], colorimetry[17]. STIP1 was recently identified as a potential tumor marker for human ovarian cancer, for which electrochemical sensor [18] has been developed to monitor its concentration. In these detection articles, CA125 and STIP1 were individually detected using antibody assays against the cancer antigen. Aptamers rival antibodies, the traditional recognition molecule, aptamers for molecular recognition addresses several of these issues: aptamers possess a low molecular weight, exhibit easy and reproducible synthesis, easy modification [19], fast tissue penetration, low toxicity or immunogenicity [20, 21], easy storage, and high binding affinity and selectivity [22, 23]. However, there were no successful reports about STIP1 aptamer until a novel aptamer TOV6 for the STIP1 molecule was reported by Tan’s group recently [24]. There were also no successful reports about CA125 aptamer until DNA strands for CA125 molecule were selected through a selective process called SELEX [25]. Hence, developing an aptamer sensor that used to detect tumor marker CA125 and STIP1 remain as unmet challenges. In this work, we successfully developed an aptamer sensor (Scheme 1B) to simultaneous quantify the concentration of CA125 and STIP1 by using RLS technique, which provide robust evidences for the presence of ovarian cancer in the
early stage. To demonstrate the stringency of this RLS-based aptamer sensor for the simultaneous detection of CA125 and STIP1, we conducted a fluorescent sensor (Scheme 1A) for the assay of the specificity of aptamers that relies on the binding of RGO to aptamer, thereby quenching the fluorescence of fluorophores that are conjugated to aptamer. The results show that these sensors exhibit desired sensitivity and specificity toward tumor markers CA125 and STIP1. Hence, this work achieves simultaneous detection of CA125 and STIP1 which provide robust evidences for the presence of ovarian cancer in the early stage. Furthermore, the successful use of aptamer in the detection of CA125 and STIP1 supports the exploration of other aptamer-based assays. 2. Experimental section 2.1. Materials and apparatus STIP1 was purchased from Abnova (Taipei city, Taiwan). CA125 was purchased from Fitzgerald, Inc. (Acton, MA, USA). DNA oligonucleotides were purchased from Sangon Biotech Co., Ltd (Shanghai China). Milli-Q deionized water was used throughout the experimental process and to prepare PBS buffers. All fluorescence and RLS measurements were carried out on a RF-5301PC fluorescence spectrophotometer (Shimadzu, Japan). AFM image was taken on a Multimode V microscope (VEECO, Inc., USA). FT-IR spectra were recorded on a Nicolet 6700 Fourier transform infrared spectrometer (Thermo Fisher Scientific, USA). UV-vis spectra were measured with a Shimadzu UV-2450 spectrophotometer. 2.2. Synthesis and functionalization of GO
GO was prepared by the Hummer’s method [26]. RGO was synthesized by the modified Li’s method [27, 28] through a reduction of GO. 0.5 g of L-alanine was added into 300 mL NaOH aqueous solution (0.1 mol⋅L-1). The resulting solution was regulated pH value to 9.6 with HCl aqueous solution. 100 mg of GO powder was added into the above solution to prepare the mixture. After ultrasonic treatment, the mixture was stirred in an oil bath at 95 °C for 4 h. Then, the mixture was centrifuged at 10000 rpm for 20 min. The sediment was washed with ultrapure water and ethanol several times. Hence, the highly reduced RGO was obtained. 2.3. Separate detection of CA125 and STIP1 based on fluorescence sensor The RGO solution was obtained by adding 10 μL (1 mg⋅mL-1) RGO into the PBS solution (10 mmol⋅L-1, pH 7.4). FAM-labeled CA125 aptamer (30μL, 100 nmol⋅L-1; 5'-TAT CAA TTA CTT ACC CTA GTG GTG TGA TGT CGT ATG GAT G-FAM-3') and Cy5-modified STIP1 aptamer (30 μL, 100 nmol⋅L-1; 5’-Cy5-CGG CAC TCA CTC TTT GTT AAG TGG TCT GCT TCT TAA CCT TCA–3’) were added into the RGO solution, the reaction was carried on for 10 min. Then, CA125 or STIP1 at different concentrations was added into the above solution, the reaction was allowed to process at 37 °C for 30 min. The fluorescence intensity was measured after the reaction. 2.4. Simultaneous detection of CA125 and STIP1 by RLS sensor CA125 aptamer (30 μL, 10 nmol⋅L-1; 5’-CGG CAC TCA CTC TTT GTT AAG TGG TCT GCT TCT TAA CCT TCA TAT CAA TTA CTT ACC CTA GTG GTG TGA TGT CGT ATG GAT G-3') and STIP1 aptamer (30 μL, 10 nmol⋅L-1; 5'-CAT
CCA TAC GAC ATC ACA CCA CTA GGG TAA GTA ATT GAT ATG AAG GTT AAG AAG CAG ACC ACT TAA CAA AGA GTG AGT GCC G-3') were added into PBS solution (10 mmol⋅L-1, pH 7.4), and the DNA hybridization reaction was conducted at 37 °C for 40 min. 30 μL (2 μmol⋅mL-1) methyl violet was subsequently added into the above solution. After 5 min of incubation, different concentrations of CA125 and STIP1 were added into the above solution. After incubation at 37 °C for 30 min, the RLS intensity of the resulting solutions was measured by synchronous scanning the excitation and emission monochromators from 200 to 700 nm. 3. Results and discussion 3.1. Design for respective and simultaneous detection of CA125 and STIP1 Scheme 1A outlined the principles for the respective detection of CA125 and STIP1 by the aptamer-based fluorescent sensor. FAM-labeled CA125 aptamer and Cy5-modified STIP1 aptamer were introduced to generate multiple signals corresponding to different targets. Respective detections of CA125 and STIP1 were accomplished in a similar procedure: in the first step, either the FAM-labeled CA125 aptamer or the Cy5-modified STIP1 aptamer was mixed with RGO so that the aptamer could adsorb on the surface of RGO through π-π stacking interaction, which lead to substantial fluorescence quenching of the FAM or Cy5 through electron transfer between fluorophore and RGO. In the second step, upon the addition of target, the aptamer changed into stable and internal loop structures. The weak binding ability of the loop-structured assembly of aptamer/target to RGO made fluorophore far away from the quencher surface, leading to fluorescence recovery of FAM or Cy5. Thus, the
corresponding fluorescence signal change could be used to respectively detect CA125 and STIP1. Scheme 1B showed the principles for the simultaneous detection of CA125 and STIP1 by the aptamer-based RLS sensor. According to the principle of our previously reported RLS sensor for simultaneous detection of lysozyme and ATP[29], CA125 aptamer firstly hybridized with STIP1 aptamer to form the dsDNA. Then, methyl violet interacted with dsDNA to amplify the RLS signals. When the mixture of CA125 and STIP1 was added, CA125 and STIP1 respectively bind to the corresponding aptamer, resulted in the formation of aptamer/target complex. These formed aptamer/target complex could change the RLS intensity. The changed RLS intensity in two different detected wavelengths met two binary function relations, which could be used to calculate the concentration of CA125 and STIP1. 3.2. The formation of RGO sheet Compared with chemically inert graphene, RGO being of active sites and function groups has been widely applications. Hence, in this work, RGO was used as an effective quencher to build the fluorescence sensor. To confirm the successful formation of a single-layered GO sheet, the prepared GO was analyzed by atomic force microscopy (AFM). As shown in Fig. 1A and B, the sheet width and height of GO were approximately 1 μm and 1 nm, respectively. The chemical structure of GO was characterized by FT-IR spectroscopy. Several characteristic peaks of functional groups containing oxygen were observed in the FT-IR spectrum of GO, including peaks at 1731 and 1043 cm-1 that resulted from C=O and C−O stretching, respectively.
Taken together, these data support the premise that single-layered GO sheets were successfully prepared [30, 31]. FT-IR spectrum and UV-Vis spectrum of GO and RGO were investigated to further illustrate the reduction of GO. In Fig. 1C, FT-IR spectrum of RGO presented that alkoxy groups were significantly decreased. In Fig. 1D, the absorption peak of the GO at 226 nm red-shifted to 251 nm and the absorption in the spectral region also increased, suggesting that the electronic conjugation within the GO sheets was restored upon a high degree of reduction. The inset of Fig. 1D showed photo for the vials of GO and RGO dispersion, after the reduction, the color of GO solution (yellow brown) changed to that of RGO solution (black). 3.3. Detection capability for STIP1 To improve the sensitivity of detection, a series of control experiments were designed to optimize the incubation time and the amount of RGO (in supplementary information). Fig. 2 illustrated that the fluorescence intensity was gradually increased with the continuous addition of STIP1, and the fluorescence intensity showed a linear relationship with the concentration of STIP1 from 1 to 75 ng⋅mL-1. The linear fitting equation could be expressed as F – F0 = 9.2851 C + 56.1488 (F represents the fluorescence intensities in the presence of STIP1; F0 represents the fluorescence intensities in the absence of STIP1; C represents the concentration of STIP1), and the corresponding correlation coefficient (R2) of the calibration curve was 0.9904. 3.4. Detection capability for CA125 In the second step in the detection of CA125, the fluorescence intensity increased
with the addition of the different CA125 concentrations shown in Fig. S1. One can notice that the minimum detectable concentration of CA125 was only 50 U⋅mL-1, which was not sufficient to detect levels of CA125 that correlate with the presence of disease (35 U⋅mL-1). 3.5. Enhance sensitivity of CA125 by formaldehyde To improve the sensitivity for the detection of CA125, we proposed a chemical fixation to improve the affinity between CA125 and its aptamer via formaldehyde. Formaldehyde is a well-known reagent, used in the study of the interaction between DNA and protein [32, 33]. In a 5% formaldehyde/PBS solution, the minimum detectable concentration of CA125 was down to 0.05 U⋅mL-1, more than thousand times of PBS solution. As shown in Fig. 3, from the range of 0.05 to 2 U⋅mL-1 of CA125, the decreased fluorescence intensity was proportional to the concentration of CA125, the linear fitting equation was F0 – F = 49.8308 C + 44.9795 (F0 represents the fluorescence intensities in the absence of CA125; F represents the fluorescence intensities in the presence of CA125; C represents the concentration of CA125), and the corresponding correlation coefficient (R2) of the calibration curve was 0.9907. 3.6. Mechanism of CA125 detection under the effect of formaldehyde It should be noted that the fluorescence intensity decreased with the increased amount of CA125 from the range of 0.05 to 2 U⋅mL-1. This may because the single-stranded DNA, the FAM-labeled CA125 aptamer was cross-linked under the effect of formaldehyde, therefore DNA duplexes was formed [34]. The DNA duplexes exhibited moderate fluorescence because the weak interaction between RGO and
double-stranded DNA. Addition of CA125 to the system separated the DNA duplexes and folds the aptamer into its stable aptamer-CA125 complex. Thus, the surplus aptamers can be adsorbed on the surface of RGO, which resulted in fluorescence quenching (shown in Scheme 2). To verify the mechanism in scheme 2, a series of experiments were carried out. The results are shown in Fig. 4. High fluorescence intensity was observed when FAM-labeled CA125 aptamer was presented alone (curve a). The fluorescence intensity sharply decreased when FAM-labeled CA125 aptamer mixed with RGO (curve b). The presence of formaldehyde recovered the fluorescence intensity to a relatively high intensity (curve c) while decrease little after CA125 was added (curve d). Further support that confirms the formation of DNA duplexes by formaldehyde was obtained by the agarose gel electrophoresis analysis. As shown in the inset of Fig. 4, in the absence of formaldehyde, a UV band of CA125 aptamer is observed (Lane 1). However, once formaldehyde is added, it binds with the aptamer, leading to the formation of DNA duplexes. These DNA duplexes with high molecular weight exhibit a clear band above that of the CA125 aptamer (Lane 2). 3.7. Specificity analysis Next, we analysed the cross-reactivity between proteins and noncognate aptamers. The solution of each protein was analysed in the absence and in the presence of the other. Each protein produced a high signal only when combined with the respective aptamer and no cross-reaction signal occurred with the other protein (Fig. 5). These results proved that the aptamer-based sensor could specifically detect target molecules.
Meanwhile, the detection of CA125 together with STIP1 also proved the aptamer-based assay could be used for simultaneous detection of CA125 and STIP1 without the interference between each other. The influences of non-specific proteins were also tested to assess the specificity of aptamer-based assay. Fig. 5 show that Alpha Fetal Protein (AFP), Prostate Specific Antigen (PSA) and thrombin, all at concentrations of 40 μg mL-1, did not caused obvious changes in the fluorescence. This result has thus clearly demonstrated that the aptamer-based assay has good specificity for the detection of CA125 and STIP1. 3.8. Simultaneous detection of CA125 and STIP1 Finally, we achieved the purpose of simultaneous detection of CA125 and STIP1 by aptamer-based RLS sensor (Scheme 1B). To clarify the effect of CA125 and STIP1 in RLS intensity at different wavelength, we showed a couple of curves with 0 CA125 and 1 ng⋅mL-1 STIP1 (curve (a) in Fig. S4), and 0.1 U⋅mL-1 CA125 and 0 STIP1 (curve (b) in Fig. S4). From curve (a) and (b), it can be found that when CA125 or STIP1 was added to RLS sensor, the RLS signal at 419 nm and 469 nm were changed together. These results show that the RLS intensity at 419 nm and 469 nm were changed by both of CA125 and STIP1. Fig. 6 showed that the intensity of the RLS signals increased with the increase of the CA125 and STIP1 concentration. With the concentration of CA125 in the range from 0.1 to 2 U⋅mL-1 and STIP1 in the range from 1 to 40 ng⋅mL-1, the changed RLS intensity at 419 nm was conformed to the relation (Fig. 6 A): ΔIRLS = 403.4314CCA125 − 9.6294CSTIP1 + 37.1625 (R=0.9812), the changed RLS intensity at 469 nm was conformed to the relation (Fig. 6 B): ΔIRLS =
539.17CCA125 − 13.63CSTIP1 + 34.383 (R=0.9845). The unique solution of the system of linear equations corresponded to the concentration of CA125 and STIP1, respectively. 3.9. Simultaneous detection of CA125 and STIP1 in serum sample Simultaneous detection of CA125 and STIP1 was performed in human serum systems. Table 1 shows that the concentration of CA125 and STIP1 can be simultaneously quantitated by the proposed RLS sensor in human serum. The recovery was calculated to further validate the assay with three different concentrations of CA125 and STIP1. The recoveries were between 93 and 107% which are very well within the acceptable range[35]. Therefore, our assay has potential application in clinical diagnostics for simultaneous detection of CA125 and STIP1. Table 1 Detection of CA125 and STIP1 in serum sample CA125
STIP1
content
STIP1 content
ΔIRLS
ΔIRLS
CA125 content
content
added
added (ng⋅mL-1)
(419nm)
(469nm)
detected (U⋅mL-1)
detected
(U⋅mL-1)
(ng⋅mL-1)
0.5
20
48.31
35.06
0.47
18.69
0.5
10
144.67
170.31
0.51
10.18
1.0
10
346.95
440.21
1.02
10.76
4. Conclusion In summary, the present study demonstrated the novel view of the early diagnosis of ovarian cancer, which is simultaneous detecting tumor marker CA125 and STIP1 by aptamer-based fluorescent and RLS sensors. The fluorescent sensor can
respectively detect CA125 and STIP1 with the lowest detectable concentration down to 0.05 U⋅mL-1 and 1 ng⋅mL-1, which falls in the expected range for clinical use of detecting disease. In addition, the RLS sensor can simultaneously detect CA125 and STIP1 in mixed solution. The simultaneous quantification of CA125 and STIP1 provide robust evidences for the presence of ovarian cancer in the early stage. Importantly, aptamer is first used to detect CA125 and STIP1with high specificity and selectivity. Thus, our study extended the application of aptamer in the detection of CA125 and STIP1. And also, we believe that the aptamer-based RLS sensor presented here will provide a valuable reference for the screening of ovarian cancer. Acknowledgment This work was supported by the National Natural Science Foundation of China (no. 21305118, 21402168, 21505112); Hunan 2011 Collaborative Innovation Center of Chemical Engineering & Technology with Environmental Benignity and Effective Resource Utilization; and Scientific Research Foundation of Hunan Provincial Education Department (No. 16A204).
References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2015, CA-Cancer J. Clin., 65 (2015) 5-29. [2] Y. Wang, R. Wu, K.R. Cho, D.G. Thomas, G. Gossner, J.R. Liu, T.J. Giordano, K.A. Shedden, D.E. Misek, D.M. Lubman, Differential Protein Mapping of Ovarian Serous Adenocarcinomas: Identification of Potential Markers for Distinct Tumor Stage, J. Proteome Res., 8 (2009) 1452-1463. [3] R.C. Bast, Jr., M. Feeney, H. Lazarus, L.M. Nadler, R.B. Colvin, R.C. Knapp, Reactivity of a monoclonal antibody with human ovarian carcinoma, J. Clin. Invest., 68 (1981) 1331-1337. [4] B. Van Calster, D. Timmerman, T. Bourne, A.C. Testa, C. Van Holsbeke, E. Domali, D. Jurkovic, P. Neven, S. Van Huffel, L. Valentin, Discrimination between benign and malignant adnexal masses by specialist ultrasound examination versus serum CA-125, J. Natl. Cancer Inst., 99 (2007) 1706-1714. [5] D.L. Clarke-Pearson, Screening for Ovarian Cancer, New Engl. J. Med., 361 (2009) 170-177. [6] I. Jacobs, R.C. Bast, The CA 125 tumour-associated antigen: a review of the literature, Hum. Reprod., 4 (1989) 1-12. [7] H. Nagata, K. Takahashi, Y. Yamane, K. Yoshino, T. Shibukawa, M. Kitao, Abnormally High Values of CA 125 and CA 19-9 in Women with Benign Tumors, Gynecol. Obstet. Inves., 28 (1989) 165-168.
[8] J. Trapé, R. Molina, F. Sant, Clinical Evaluation of the Simultaneous Determination of Tumor Markers in Fluid and Serum and Their Ratio in the Differential Diagnosis of Serous Effusions, Tumor Biol., 25 (2004) 276-281. [9] R. Molina, X. Filella, J. Bruix, P. Mengual, J. Bosch, X. Calvet, J. Jo, A.M. Ballesta, Cancer antigen 125 in serum and ascitic fluid of patients with liver diseases, Clin. Chem., 37 (1991) 1379-1383. [10] J. Trape, X. Filella, M. Alsina-Donadeu, L. Juan-Pereira, A. Bosch-Ferrer, R. Rigo-Bonnin, Increased plasma concentrations of tumour markers in the absence of neoplasia, Clin. Chem. Lab. Med., 49 (2011) 1605-1620. [11] T.H. Wang, A. Chao, C.L. Tsai, C.L. Chang, S.H. Chen, Y.S. Lee, J.K. Chen, Y.J. Lin, P.Y. Chang, C.J. Wang, A.S. Chao, S.D. Chang, T.C. Chang, C.H. Lai, H.S. Wang, Stress-induced phosphoprotein 1 as a secreted biomarker for human ovarian cancer promotes cancer cell proliferation, Mol. Cell Proteomics, 9 (2010) 1873-1884. [12] S.A. McQuarrie, R.P. Baum, A. Niesen, R. Madiyalakan, W. Korz, T.R. Sykes, C.J. Sykes, G. Hör, A.J.B. McEwan, A.A. Noujaim, Pharmacokinetics and radiation dosimetry of 99Tcm-labelled monoclonal antibody B43.13 in ovarian cancer patients, Nucl. Med. Commun., 18 (1997) 878-886. [13] G. Yan, H. Ju, Z. Liang, T. Zhang, Technical and clinical comparison of two fully automated methods for the immunoassay of CA 125 in serum, J. Immunol. Methods, 225 (1999) 1-8. [14] S. Suwansa-ard, P. Kanatharana, P. Asawatreratanakul, B. Wongkittisuksa, C. Limsakul, P. Thavarungkul, Comparison of surface plasmon resonance and capacitive
immunosensors for cancer antigen 125 detection in human serum samples, Biosens. Bioelectron., 24 (2009) 3436-3441. [15] M. Johari-Ahar, M.R. Rashidi, J. Barar, M. Aghaie, D. Mohammadnejad, A. Ramazani, P. Karami, G. Coukos, Y. Omidi, An ultra-sensitive impedimetric immunosensor for detection of the serum oncomarker CA-125 in ovarian cancer patients, Nanoscale, 7 (2015) 3768-3779. [16] J. Das, S.O. Kelley, Protein Detection Using Arrayed Microsensor Chips: Tuning Sensor Footprint to Achieve Ultrasensitive Readout of CA-125 in Serum and Whole Blood, Anal. Chem., 83 (2011) 1167-1172. [17] Y. Zhao, Y. Zheng, C. Zhao, J. You, F. Qu, Hollow PDA-Au nanoparticles-enabled signal amplification for sensitive nonenzymatic colorimetric immunodetection of carbohydrate antigen 125, Biosens. Bioelectron., 71 (2015) 200-206. [18] J. Lee, S. Cho, J. Lee, H. Ryu, J. Park, S. Lim, B. Oh, C. Lee, W. Huang, A. Busnaina, H. Lee, Wafer-scale nanowell array patterning based electrochemical impedimetric immunosensor, J. Biotechnol., 168 (2013) 584-588. [19] J.J. Li, X. Fang, W. Tan, Molecular Aptamer Beacons for Real-Time Protein Recognition, Biochem. Bioph. Res. Commun., 292 (2002) 31-40. [20] K.K. Knox, J.H. Brewer, J.M. Henry, D.J. Harrington, D.R. Carrigan, Human Herpesvirus 6 and Multiple Sclerosis: Systemic Active Infections in Patients with Early Disease, Clin. Infect. Dis., 31 (2000) 894-903. [21] L. Cerchia, J. Hamm, D. Libri, B. Tavitian, V. de Franciscis, Nucleic acid
aptamers in cancer medicine, Febs Lett., 528 (2002) 12-16. [22] L. Gold, Oligonucleotides as research, diagnostic, and therapeutic agents, J. Biol. Chem., 270 (1995) 13581-13584. [23] T. Hermann, D.J. Patel, Adaptive Recognition by Nucleic Acid Aptamers, Science, 287 (2000) 820-825. [24] D. Van Simaeys, D. Turek, C. Champanhac, J. Vaizer, K. Sefah, J. Zhen, R. Sutphen, W. Tan, Identification of Cell Membrane Protein Stress-Induced Phosphoprotein 1 as a Potential Ovarian Cancer Biomarker Using Aptamers Selected by Cell Systematic Evolution of Ligands by Exponential Enrichment, Anal. Chem., 86 (2014) 4521-4527. [25] A.M. Whited, Investigation of Impedance Spectroscopy for Detection of Ovarian Cancer, (2012). [26] W.S. Hummers, R.E. Offeman, Preparation of Graphitic Oxide, J. Am. Chem. Soc., 80 (1958) 1339-1339. [27] D. Li, M.B. Muller, S. Gilje, R.B. Kaner, G.G. Wallace, Processable aqueous dispersions of graphene nanosheets, Nat. Nanotechnol., 3 (2008) 101-105. [28] K. He, R. Liao, C. Cai, C. Liang, C. Liu, X. Chen, Y-shaped probe for convenient and label-free detection of microRNA-21 in vitro, Anal. Biochem., 499 (2016) 8-14. [29] F. Chen, C. Cai, X. Chen, C. Chen, “Click on the bidirectional switch”: the aptasensor for simultaneous detection of lysozyme and ATP with high sensitivity and high selectivity, Sci. Rep., 6 (2016) 18814. [30] H. Jang, Y.-K. Kim, H.-M. Kwon, W.-S. Yeo, D.-E. Kim, D.-H. Min, A
Graphene-Based Platform for the Assay of Duplex-DNA Unwinding by Helicase, Angew. Chem. Int. Ed., 122 (2010) 5839-5843. [31] H. Jang, S.-R. Ryoo, Y.-K. Kim, S. Yoon, H. Kim, S.W. Han, B.-S. Choi, D.-E. Kim, D.-H. Min, Discovery of Hepatitis C Virus NS3 Helicase Inhibitors by a Multiplexed, High-Throughput Helicase Activity Assay Based on Graphene Oxide, Angew. Chem. Int. Ed., 52 (2013) 2340-2344. [32]
M.J.
Solomon,
A.
Varshavsky,
Formaldehyde-mediated
DNA-protein
crosslinking: a probe for in vivo chromatin structures, P. Natl. Acad. Sci. USA, 82 (1985) 6470-6474. [33] J. Kennedy-Darling, L.M. Smith, Measuring the Formaldehyde Protein–DNA Cross-Link Reversal Rate, Anal. Chem., 86 (2014) 5678-5681. [34] J.D. McGhee, P.H. Von Hippel, Formaldehyde as a probe of DNA structure. I. Reaction with exocyclic amino groups of DNA bases, Biochemistry, 14 (1975) 1281-1296. [35] I. Taverniers, M. De Loose, E. Van Bockstaele, Trends in quality in the analytical laboratory. II. Analytical method validation and quality assurance, TrAC-Trend. Anal. Chem., 23 (2004) 535-552.
Biographies
Changqun Cai received her Ph.D in the college of chemistry from Xiangtan University in 2011, Xiangtan, Hunan, China. She holds the position of Associate Professor in Analytical Chemistry at College of Chemistry, Xiangtan University. Her research interests focus on Analytical Chemistry in Life Science and Biosensor. Hang Gong received his Ph.D in the college of chemistry from Xiangtan University in 2011, Xiangtan, Hunan, China. He worked as a faculty in College of Chemistry, Xiangtan University. His research interests focus on Analytical Chemistry in Life Science and organic synthetic methodology. Feng Chen is a M.S. student at Xiangtan University. Her research interests include spectral analysis and biosensor. Yi Liu is a M.S. student at Xiangtan University. Her research interests include spectral analysis and biosensor. Chunyan Chen received her Ph.D. in 2013 from Hunan University, Shangcha, Hunan, China. She worked as a faculty in College of Chemistry, Xiangtan University. She current research focus is chromatographic analysis. Xiaoming Chen is a professor of analytical chemistry in Xiangtan University, His research interests are chromatographic analysis and optical analysis.
Fig. 1. (A) AFM image and (B) height profile of GO, (C) FT-IR spectra of GO and RGO, (D) UV-Vis absorption spectra of GO and RGO in water
Fig. 2 Fluorescence spectra and variance of normalized fluorescence intensity with the concentration of STIP1: (1) 0, (2) 1, (3) 5, (4) 25, (5) 50, (6) 75 ng⋅mL-1. Excitation wavelength was 625 nm.
Fig. 3 Fluorescence spectra and variance of normalized fluorescence intensity with the concentration of CA125: (1) 0, (2) 0.05, (3) 0.1, (4) 0.5, (5) 1, (6) 2 U⋅mL-1. Excitation wavelength was 490 nm.
Fig. 4 Fluorescence responses of FAM-labeled CA125 aptamer (a), FAM-labeled CA125 aptamer + RGO (b), FAM-labeled CA125 aptamer + RGO + formaldehyde (c), FAM-labeled CA125 aptamer + RGO + formaldehyde + CA125 (d). Excitation wavelength was 490 nm. Inset: the DNA duplexes were analysed using agarose gel electrophoresis, (Lane1) FAM-labeled CA125 aptamer, (lane2) the mixture in FAM-labeled CA125 aptamer and formaldehyde.
Fig. 5 Study of the specificity of aptamers for the detection of CA125 and STIP1 (100 U⋅mL-1 for CA125; 1 ng⋅mL-1 for STIP1; 40 μg mL-1 for non-specific proteins).
A
B
Fig. 6 RLS spectra and the changed RLS intensity with the concentration of mixtures of CA125 and STIP1. (A) The changed RLS intensity at 419 nm. (B) The changed RLS intensity at 469 nm. Conditions: different concentrations of CA125: (1) 0, (2) 0.1, (3) 0.2, (4) 0.4, (5) 0.6, (6) 0.8, (7)1, (8) 2 U⋅mL-1; different concentrations of STIP1: (1) 0, (2) 1, (3) 2, (4) 4, (5) 8, (6) 10, (7) 20, (8) 40 ng⋅mL-1.
Scheme 1. Schematic representation of the assay of CA125 and STIP1 using aptamer-based fluorescent sensor (A) and RLS sensor (B).
Scheme 2 Schematic representation of the assay of CA125 via formaldehyde cross-linking.
S0925-4005(17)30162-4 http://dx.doi.org/doi:10.1016/j.snb.2017.01.155 SNB 21680
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
6-12-2016 16-1-2017 24-1-2017
Please cite this article as: Feng Chen, Yi Liu, Chunyan Chen, Hang Gong, Changqun Cai, Xiaoming Chen, Respective and simultaneous detection tumor markers CA125 and STIP1 using aptamer-based fluorescent and RLS sensors, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2017.01.155 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Respective and simultaneous detection tumor markers CA125 and STIP1 using aptamer-based fluorescent and RLS sensors Feng Chen, Yi Liu, Chunyan Chen, Hang Gong*, Changqun Cai*, Xiaoming Chen Key Laboratory of Environmentally Friendly Chemistry and Applications of Ministry of Education, College of Chemistry, Xiangtan University, Xiangtan, Hunan 411105, China. Corresponding author. Tel: +86 152 7321 9560. Fax: +86-732-5829-2251. Email: [email protected] (H.Gong); [email protected] (C.Cai).
Graphical abstract:
Tumor marker CA125 and STIP1 were respectively and simultaneously detected using aptamers with fluorescence based detection on RGO as well as RLS detection for screening ovarian cancer.
Highlighs:
A novel strategy for screening ovarian cancer is proposed.
The strategy is individual and simultaneous detection of tumor marker CA125 and STIP1 using fluorescence intensity and resonance light scattering.
The detection for CA125 and STIP1 is using aptamer as binding element for the first time.
Abstract Here we reported a method for the early diagnosis of ovarian cancer that is respective and simultaneous detection of tumour marker cancer antigen 125 (CA125) and stress-induced phosphoprotein 1 (STIP1) using aptamer-based fluorescent and RLS sensors. The fluorescent sensor was fabricated by Carboxyfluorescein (FAM)-labelled CA125 aptamer and cyanine-5-modified STIP1 aptamer onto the surface of RGO, thereby quenching the fluorescence of fluorophores. When STIP1 was introduced, the fluorescence recovered. When CA125 was added, the specific binding of aptamer and CA125 triggered the release of the FAM-labeled CA125 aptamer under the effect of formaldehyde, and adsorbed on the surface of RGO which lead to fluorescence quenching of FAM. This fluorescence signals change could be used to detect CA125 and STIP1 with the lowest detectable concentration down to 0.05 U⋅mL-1 and 1 ng⋅mL-1 respectively. The RLS sensor was fabricated by methyl violet interact with dsDNA which is obtained by CA125 aptamer hybridized to STIP1 aptamer. The dependence of RLS intensity on targets amount was successfully utilized for simultaneous detection of CA125 and STIP1, which provide robust evidences for the presence of ovarian cancer in the early stage. This strategy is reliable, sensitive, and may form the basis for rapid screening of ovarian cancer. Keywords: Aptamer; Resonance light scattering sensor; Early diagnosis of ovarian cancer; Cancer antigen 125; Stress-induced phosphoprotein 1
1. Introduction Ovarian cancer is the leading cause of gynaecological cancer death [1]. The high case fatality rate is mainly attributed to its nonspecific symptoms and lack of an adequate physical exam; over 75 percent of cases are diagnosed in the late stages when the disease has metastasized beyond the primary site, leading to a five year survival rate of less than 30 percent [2]. Since the first characterization of a monoclonal antibody against a glycoprotein, CA125, in 1981 [3], measurement of serum levels of CA125 has become standard practice for the preoperative evaluation of ovarian masses [4]. However, the sensitivity of CA125 is only about 80% for detecting overall ovarian cancer [5] and only about 50% for early stage cancer [6]. In clinical trials, most abnormal CA125 values are false positives that may be caused by benign ovarian disease [7] or benign fluid effusion [8] or hepatopathies with ascites [9] or Meigs’syndrome [10]. These findings suggest that CA125 alone is not an appropriate surrogate marker for the diagnosis and screening of ovarian cancer. Wang et al. reported that STIP1 was secreted by ovarian cancer tissues into the peripheral blood of patients and the combined measurement of CA125 and STIP1 has been shown to improve the early detection of ovarian cancer [11]. The combined use of CA125﹥35 U⋅mL-1 and STIP1﹥55 ng⋅mL-1 in detecting ovarian cancer, resulted in 100% sensitivity, 95.3% specificity, 95.6% positive predictive value, and 100% negative predictive value. Hence, it is desired to achieve simultaneous detection of CA125 and STIP1 which provide robust evidences for the presence of ovarian cancer in the early stage.
CA125 plays vital role in the diagnosis of ovarian cancer. For clinical laboratory, CA125 levels are usually measured by radiometric[12] and enzyme immunoassay[13]. These conventional assays are time-consuming and required special equipped laboratories. In recent decades, several assays have been developed to detect CA125, including surface plasmon resonance[14], electrochemistry[15, 16], colorimetry[17]. STIP1 was recently identified as a potential tumor marker for human ovarian cancer, for which electrochemical sensor [18] has been developed to monitor its concentration. In these detection articles, CA125 and STIP1 were individually detected using antibody assays against the cancer antigen. Aptamers rival antibodies, the traditional recognition molecule, aptamers for molecular recognition addresses several of these issues: aptamers possess a low molecular weight, exhibit easy and reproducible synthesis, easy modification [19], fast tissue penetration, low toxicity or immunogenicity [20, 21], easy storage, and high binding affinity and selectivity [22, 23]. However, there were no successful reports about STIP1 aptamer until a novel aptamer TOV6 for the STIP1 molecule was reported by Tan’s group recently [24]. There were also no successful reports about CA125 aptamer until DNA strands for CA125 molecule were selected through a selective process called SELEX [25]. Hence, developing an aptamer sensor that used to detect tumor marker CA125 and STIP1 remain as unmet challenges. In this work, we successfully developed an aptamer sensor (Scheme 1B) to simultaneous quantify the concentration of CA125 and STIP1 by using RLS technique, which provide robust evidences for the presence of ovarian cancer in the
early stage. To demonstrate the stringency of this RLS-based aptamer sensor for the simultaneous detection of CA125 and STIP1, we conducted a fluorescent sensor (Scheme 1A) for the assay of the specificity of aptamers that relies on the binding of RGO to aptamer, thereby quenching the fluorescence of fluorophores that are conjugated to aptamer. The results show that these sensors exhibit desired sensitivity and specificity toward tumor markers CA125 and STIP1. Hence, this work achieves simultaneous detection of CA125 and STIP1 which provide robust evidences for the presence of ovarian cancer in the early stage. Furthermore, the successful use of aptamer in the detection of CA125 and STIP1 supports the exploration of other aptamer-based assays. 2. Experimental section 2.1. Materials and apparatus STIP1 was purchased from Abnova (Taipei city, Taiwan). CA125 was purchased from Fitzgerald, Inc. (Acton, MA, USA). DNA oligonucleotides were purchased from Sangon Biotech Co., Ltd (Shanghai China). Milli-Q deionized water was used throughout the experimental process and to prepare PBS buffers. All fluorescence and RLS measurements were carried out on a RF-5301PC fluorescence spectrophotometer (Shimadzu, Japan). AFM image was taken on a Multimode V microscope (VEECO, Inc., USA). FT-IR spectra were recorded on a Nicolet 6700 Fourier transform infrared spectrometer (Thermo Fisher Scientific, USA). UV-vis spectra were measured with a Shimadzu UV-2450 spectrophotometer. 2.2. Synthesis and functionalization of GO
GO was prepared by the Hummer’s method [26]. RGO was synthesized by the modified Li’s method [27, 28] through a reduction of GO. 0.5 g of L-alanine was added into 300 mL NaOH aqueous solution (0.1 mol⋅L-1). The resulting solution was regulated pH value to 9.6 with HCl aqueous solution. 100 mg of GO powder was added into the above solution to prepare the mixture. After ultrasonic treatment, the mixture was stirred in an oil bath at 95 °C for 4 h. Then, the mixture was centrifuged at 10000 rpm for 20 min. The sediment was washed with ultrapure water and ethanol several times. Hence, the highly reduced RGO was obtained. 2.3. Separate detection of CA125 and STIP1 based on fluorescence sensor The RGO solution was obtained by adding 10 μL (1 mg⋅mL-1) RGO into the PBS solution (10 mmol⋅L-1, pH 7.4). FAM-labeled CA125 aptamer (30μL, 100 nmol⋅L-1; 5'-TAT CAA TTA CTT ACC CTA GTG GTG TGA TGT CGT ATG GAT G-FAM-3') and Cy5-modified STIP1 aptamer (30 μL, 100 nmol⋅L-1; 5’-Cy5-CGG CAC TCA CTC TTT GTT AAG TGG TCT GCT TCT TAA CCT TCA–3’) were added into the RGO solution, the reaction was carried on for 10 min. Then, CA125 or STIP1 at different concentrations was added into the above solution, the reaction was allowed to process at 37 °C for 30 min. The fluorescence intensity was measured after the reaction. 2.4. Simultaneous detection of CA125 and STIP1 by RLS sensor CA125 aptamer (30 μL, 10 nmol⋅L-1; 5’-CGG CAC TCA CTC TTT GTT AAG TGG TCT GCT TCT TAA CCT TCA TAT CAA TTA CTT ACC CTA GTG GTG TGA TGT CGT ATG GAT G-3') and STIP1 aptamer (30 μL, 10 nmol⋅L-1; 5'-CAT
CCA TAC GAC ATC ACA CCA CTA GGG TAA GTA ATT GAT ATG AAG GTT AAG AAG CAG ACC ACT TAA CAA AGA GTG AGT GCC G-3') were added into PBS solution (10 mmol⋅L-1, pH 7.4), and the DNA hybridization reaction was conducted at 37 °C for 40 min. 30 μL (2 μmol⋅mL-1) methyl violet was subsequently added into the above solution. After 5 min of incubation, different concentrations of CA125 and STIP1 were added into the above solution. After incubation at 37 °C for 30 min, the RLS intensity of the resulting solutions was measured by synchronous scanning the excitation and emission monochromators from 200 to 700 nm. 3. Results and discussion 3.1. Design for respective and simultaneous detection of CA125 and STIP1 Scheme 1A outlined the principles for the respective detection of CA125 and STIP1 by the aptamer-based fluorescent sensor. FAM-labeled CA125 aptamer and Cy5-modified STIP1 aptamer were introduced to generate multiple signals corresponding to different targets. Respective detections of CA125 and STIP1 were accomplished in a similar procedure: in the first step, either the FAM-labeled CA125 aptamer or the Cy5-modified STIP1 aptamer was mixed with RGO so that the aptamer could adsorb on the surface of RGO through π-π stacking interaction, which lead to substantial fluorescence quenching of the FAM or Cy5 through electron transfer between fluorophore and RGO. In the second step, upon the addition of target, the aptamer changed into stable and internal loop structures. The weak binding ability of the loop-structured assembly of aptamer/target to RGO made fluorophore far away from the quencher surface, leading to fluorescence recovery of FAM or Cy5. Thus, the
corresponding fluorescence signal change could be used to respectively detect CA125 and STIP1. Scheme 1B showed the principles for the simultaneous detection of CA125 and STIP1 by the aptamer-based RLS sensor. According to the principle of our previously reported RLS sensor for simultaneous detection of lysozyme and ATP[29], CA125 aptamer firstly hybridized with STIP1 aptamer to form the dsDNA. Then, methyl violet interacted with dsDNA to amplify the RLS signals. When the mixture of CA125 and STIP1 was added, CA125 and STIP1 respectively bind to the corresponding aptamer, resulted in the formation of aptamer/target complex. These formed aptamer/target complex could change the RLS intensity. The changed RLS intensity in two different detected wavelengths met two binary function relations, which could be used to calculate the concentration of CA125 and STIP1. 3.2. The formation of RGO sheet Compared with chemically inert graphene, RGO being of active sites and function groups has been widely applications. Hence, in this work, RGO was used as an effective quencher to build the fluorescence sensor. To confirm the successful formation of a single-layered GO sheet, the prepared GO was analyzed by atomic force microscopy (AFM). As shown in Fig. 1A and B, the sheet width and height of GO were approximately 1 μm and 1 nm, respectively. The chemical structure of GO was characterized by FT-IR spectroscopy. Several characteristic peaks of functional groups containing oxygen were observed in the FT-IR spectrum of GO, including peaks at 1731 and 1043 cm-1 that resulted from C=O and C−O stretching, respectively.
Taken together, these data support the premise that single-layered GO sheets were successfully prepared [30, 31]. FT-IR spectrum and UV-Vis spectrum of GO and RGO were investigated to further illustrate the reduction of GO. In Fig. 1C, FT-IR spectrum of RGO presented that alkoxy groups were significantly decreased. In Fig. 1D, the absorption peak of the GO at 226 nm red-shifted to 251 nm and the absorption in the spectral region also increased, suggesting that the electronic conjugation within the GO sheets was restored upon a high degree of reduction. The inset of Fig. 1D showed photo for the vials of GO and RGO dispersion, after the reduction, the color of GO solution (yellow brown) changed to that of RGO solution (black). 3.3. Detection capability for STIP1 To improve the sensitivity of detection, a series of control experiments were designed to optimize the incubation time and the amount of RGO (in supplementary information). Fig. 2 illustrated that the fluorescence intensity was gradually increased with the continuous addition of STIP1, and the fluorescence intensity showed a linear relationship with the concentration of STIP1 from 1 to 75 ng⋅mL-1. The linear fitting equation could be expressed as F – F0 = 9.2851 C + 56.1488 (F represents the fluorescence intensities in the presence of STIP1; F0 represents the fluorescence intensities in the absence of STIP1; C represents the concentration of STIP1), and the corresponding correlation coefficient (R2) of the calibration curve was 0.9904. 3.4. Detection capability for CA125 In the second step in the detection of CA125, the fluorescence intensity increased
with the addition of the different CA125 concentrations shown in Fig. S1. One can notice that the minimum detectable concentration of CA125 was only 50 U⋅mL-1, which was not sufficient to detect levels of CA125 that correlate with the presence of disease (35 U⋅mL-1). 3.5. Enhance sensitivity of CA125 by formaldehyde To improve the sensitivity for the detection of CA125, we proposed a chemical fixation to improve the affinity between CA125 and its aptamer via formaldehyde. Formaldehyde is a well-known reagent, used in the study of the interaction between DNA and protein [32, 33]. In a 5% formaldehyde/PBS solution, the minimum detectable concentration of CA125 was down to 0.05 U⋅mL-1, more than thousand times of PBS solution. As shown in Fig. 3, from the range of 0.05 to 2 U⋅mL-1 of CA125, the decreased fluorescence intensity was proportional to the concentration of CA125, the linear fitting equation was F0 – F = 49.8308 C + 44.9795 (F0 represents the fluorescence intensities in the absence of CA125; F represents the fluorescence intensities in the presence of CA125; C represents the concentration of CA125), and the corresponding correlation coefficient (R2) of the calibration curve was 0.9907. 3.6. Mechanism of CA125 detection under the effect of formaldehyde It should be noted that the fluorescence intensity decreased with the increased amount of CA125 from the range of 0.05 to 2 U⋅mL-1. This may because the single-stranded DNA, the FAM-labeled CA125 aptamer was cross-linked under the effect of formaldehyde, therefore DNA duplexes was formed [34]. The DNA duplexes exhibited moderate fluorescence because the weak interaction between RGO and
double-stranded DNA. Addition of CA125 to the system separated the DNA duplexes and folds the aptamer into its stable aptamer-CA125 complex. Thus, the surplus aptamers can be adsorbed on the surface of RGO, which resulted in fluorescence quenching (shown in Scheme 2). To verify the mechanism in scheme 2, a series of experiments were carried out. The results are shown in Fig. 4. High fluorescence intensity was observed when FAM-labeled CA125 aptamer was presented alone (curve a). The fluorescence intensity sharply decreased when FAM-labeled CA125 aptamer mixed with RGO (curve b). The presence of formaldehyde recovered the fluorescence intensity to a relatively high intensity (curve c) while decrease little after CA125 was added (curve d). Further support that confirms the formation of DNA duplexes by formaldehyde was obtained by the agarose gel electrophoresis analysis. As shown in the inset of Fig. 4, in the absence of formaldehyde, a UV band of CA125 aptamer is observed (Lane 1). However, once formaldehyde is added, it binds with the aptamer, leading to the formation of DNA duplexes. These DNA duplexes with high molecular weight exhibit a clear band above that of the CA125 aptamer (Lane 2). 3.7. Specificity analysis Next, we analysed the cross-reactivity between proteins and noncognate aptamers. The solution of each protein was analysed in the absence and in the presence of the other. Each protein produced a high signal only when combined with the respective aptamer and no cross-reaction signal occurred with the other protein (Fig. 5). These results proved that the aptamer-based sensor could specifically detect target molecules.
Meanwhile, the detection of CA125 together with STIP1 also proved the aptamer-based assay could be used for simultaneous detection of CA125 and STIP1 without the interference between each other. The influences of non-specific proteins were also tested to assess the specificity of aptamer-based assay. Fig. 5 show that Alpha Fetal Protein (AFP), Prostate Specific Antigen (PSA) and thrombin, all at concentrations of 40 μg mL-1, did not caused obvious changes in the fluorescence. This result has thus clearly demonstrated that the aptamer-based assay has good specificity for the detection of CA125 and STIP1. 3.8. Simultaneous detection of CA125 and STIP1 Finally, we achieved the purpose of simultaneous detection of CA125 and STIP1 by aptamer-based RLS sensor (Scheme 1B). To clarify the effect of CA125 and STIP1 in RLS intensity at different wavelength, we showed a couple of curves with 0 CA125 and 1 ng⋅mL-1 STIP1 (curve (a) in Fig. S4), and 0.1 U⋅mL-1 CA125 and 0 STIP1 (curve (b) in Fig. S4). From curve (a) and (b), it can be found that when CA125 or STIP1 was added to RLS sensor, the RLS signal at 419 nm and 469 nm were changed together. These results show that the RLS intensity at 419 nm and 469 nm were changed by both of CA125 and STIP1. Fig. 6 showed that the intensity of the RLS signals increased with the increase of the CA125 and STIP1 concentration. With the concentration of CA125 in the range from 0.1 to 2 U⋅mL-1 and STIP1 in the range from 1 to 40 ng⋅mL-1, the changed RLS intensity at 419 nm was conformed to the relation (Fig. 6 A): ΔIRLS = 403.4314CCA125 − 9.6294CSTIP1 + 37.1625 (R=0.9812), the changed RLS intensity at 469 nm was conformed to the relation (Fig. 6 B): ΔIRLS =
539.17CCA125 − 13.63CSTIP1 + 34.383 (R=0.9845). The unique solution of the system of linear equations corresponded to the concentration of CA125 and STIP1, respectively. 3.9. Simultaneous detection of CA125 and STIP1 in serum sample Simultaneous detection of CA125 and STIP1 was performed in human serum systems. Table 1 shows that the concentration of CA125 and STIP1 can be simultaneously quantitated by the proposed RLS sensor in human serum. The recovery was calculated to further validate the assay with three different concentrations of CA125 and STIP1. The recoveries were between 93 and 107% which are very well within the acceptable range[35]. Therefore, our assay has potential application in clinical diagnostics for simultaneous detection of CA125 and STIP1. Table 1 Detection of CA125 and STIP1 in serum sample CA125
STIP1
content
STIP1 content
ΔIRLS
ΔIRLS
CA125 content
content
added
added (ng⋅mL-1)
(419nm)
(469nm)
detected (U⋅mL-1)
detected
(U⋅mL-1)
(ng⋅mL-1)
0.5
20
48.31
35.06
0.47
18.69
0.5
10
144.67
170.31
0.51
10.18
1.0
10
346.95
440.21
1.02
10.76
4. Conclusion In summary, the present study demonstrated the novel view of the early diagnosis of ovarian cancer, which is simultaneous detecting tumor marker CA125 and STIP1 by aptamer-based fluorescent and RLS sensors. The fluorescent sensor can
respectively detect CA125 and STIP1 with the lowest detectable concentration down to 0.05 U⋅mL-1 and 1 ng⋅mL-1, which falls in the expected range for clinical use of detecting disease. In addition, the RLS sensor can simultaneously detect CA125 and STIP1 in mixed solution. The simultaneous quantification of CA125 and STIP1 provide robust evidences for the presence of ovarian cancer in the early stage. Importantly, aptamer is first used to detect CA125 and STIP1with high specificity and selectivity. Thus, our study extended the application of aptamer in the detection of CA125 and STIP1. And also, we believe that the aptamer-based RLS sensor presented here will provide a valuable reference for the screening of ovarian cancer. Acknowledgment This work was supported by the National Natural Science Foundation of China (no. 21305118, 21402168, 21505112); Hunan 2011 Collaborative Innovation Center of Chemical Engineering & Technology with Environmental Benignity and Effective Resource Utilization; and Scientific Research Foundation of Hunan Provincial Education Department (No. 16A204).
References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2015, CA-Cancer J. Clin., 65 (2015) 5-29. [2] Y. Wang, R. Wu, K.R. Cho, D.G. Thomas, G. Gossner, J.R. Liu, T.J. Giordano, K.A. Shedden, D.E. Misek, D.M. Lubman, Differential Protein Mapping of Ovarian Serous Adenocarcinomas: Identification of Potential Markers for Distinct Tumor Stage, J. Proteome Res., 8 (2009) 1452-1463. [3] R.C. Bast, Jr., M. Feeney, H. Lazarus, L.M. Nadler, R.B. Colvin, R.C. Knapp, Reactivity of a monoclonal antibody with human ovarian carcinoma, J. Clin. Invest., 68 (1981) 1331-1337. [4] B. Van Calster, D. Timmerman, T. Bourne, A.C. Testa, C. Van Holsbeke, E. Domali, D. Jurkovic, P. Neven, S. Van Huffel, L. Valentin, Discrimination between benign and malignant adnexal masses by specialist ultrasound examination versus serum CA-125, J. Natl. Cancer Inst., 99 (2007) 1706-1714. [5] D.L. Clarke-Pearson, Screening for Ovarian Cancer, New Engl. J. Med., 361 (2009) 170-177. [6] I. Jacobs, R.C. Bast, The CA 125 tumour-associated antigen: a review of the literature, Hum. Reprod., 4 (1989) 1-12. [7] H. Nagata, K. Takahashi, Y. Yamane, K. Yoshino, T. Shibukawa, M. Kitao, Abnormally High Values of CA 125 and CA 19-9 in Women with Benign Tumors, Gynecol. Obstet. Inves., 28 (1989) 165-168.
[8] J. Trapé, R. Molina, F. Sant, Clinical Evaluation of the Simultaneous Determination of Tumor Markers in Fluid and Serum and Their Ratio in the Differential Diagnosis of Serous Effusions, Tumor Biol., 25 (2004) 276-281. [9] R. Molina, X. Filella, J. Bruix, P. Mengual, J. Bosch, X. Calvet, J. Jo, A.M. Ballesta, Cancer antigen 125 in serum and ascitic fluid of patients with liver diseases, Clin. Chem., 37 (1991) 1379-1383. [10] J. Trape, X. Filella, M. Alsina-Donadeu, L. Juan-Pereira, A. Bosch-Ferrer, R. Rigo-Bonnin, Increased plasma concentrations of tumour markers in the absence of neoplasia, Clin. Chem. Lab. Med., 49 (2011) 1605-1620. [11] T.H. Wang, A. Chao, C.L. Tsai, C.L. Chang, S.H. Chen, Y.S. Lee, J.K. Chen, Y.J. Lin, P.Y. Chang, C.J. Wang, A.S. Chao, S.D. Chang, T.C. Chang, C.H. Lai, H.S. Wang, Stress-induced phosphoprotein 1 as a secreted biomarker for human ovarian cancer promotes cancer cell proliferation, Mol. Cell Proteomics, 9 (2010) 1873-1884. [12] S.A. McQuarrie, R.P. Baum, A. Niesen, R. Madiyalakan, W. Korz, T.R. Sykes, C.J. Sykes, G. Hör, A.J.B. McEwan, A.A. Noujaim, Pharmacokinetics and radiation dosimetry of 99Tcm-labelled monoclonal antibody B43.13 in ovarian cancer patients, Nucl. Med. Commun., 18 (1997) 878-886. [13] G. Yan, H. Ju, Z. Liang, T. Zhang, Technical and clinical comparison of two fully automated methods for the immunoassay of CA 125 in serum, J. Immunol. Methods, 225 (1999) 1-8. [14] S. Suwansa-ard, P. Kanatharana, P. Asawatreratanakul, B. Wongkittisuksa, C. Limsakul, P. Thavarungkul, Comparison of surface plasmon resonance and capacitive
immunosensors for cancer antigen 125 detection in human serum samples, Biosens. Bioelectron., 24 (2009) 3436-3441. [15] M. Johari-Ahar, M.R. Rashidi, J. Barar, M. Aghaie, D. Mohammadnejad, A. Ramazani, P. Karami, G. Coukos, Y. Omidi, An ultra-sensitive impedimetric immunosensor for detection of the serum oncomarker CA-125 in ovarian cancer patients, Nanoscale, 7 (2015) 3768-3779. [16] J. Das, S.O. Kelley, Protein Detection Using Arrayed Microsensor Chips: Tuning Sensor Footprint to Achieve Ultrasensitive Readout of CA-125 in Serum and Whole Blood, Anal. Chem., 83 (2011) 1167-1172. [17] Y. Zhao, Y. Zheng, C. Zhao, J. You, F. Qu, Hollow PDA-Au nanoparticles-enabled signal amplification for sensitive nonenzymatic colorimetric immunodetection of carbohydrate antigen 125, Biosens. Bioelectron., 71 (2015) 200-206. [18] J. Lee, S. Cho, J. Lee, H. Ryu, J. Park, S. Lim, B. Oh, C. Lee, W. Huang, A. Busnaina, H. Lee, Wafer-scale nanowell array patterning based electrochemical impedimetric immunosensor, J. Biotechnol., 168 (2013) 584-588. [19] J.J. Li, X. Fang, W. Tan, Molecular Aptamer Beacons for Real-Time Protein Recognition, Biochem. Bioph. Res. Commun., 292 (2002) 31-40. [20] K.K. Knox, J.H. Brewer, J.M. Henry, D.J. Harrington, D.R. Carrigan, Human Herpesvirus 6 and Multiple Sclerosis: Systemic Active Infections in Patients with Early Disease, Clin. Infect. Dis., 31 (2000) 894-903. [21] L. Cerchia, J. Hamm, D. Libri, B. Tavitian, V. de Franciscis, Nucleic acid
aptamers in cancer medicine, Febs Lett., 528 (2002) 12-16. [22] L. Gold, Oligonucleotides as research, diagnostic, and therapeutic agents, J. Biol. Chem., 270 (1995) 13581-13584. [23] T. Hermann, D.J. Patel, Adaptive Recognition by Nucleic Acid Aptamers, Science, 287 (2000) 820-825. [24] D. Van Simaeys, D. Turek, C. Champanhac, J. Vaizer, K. Sefah, J. Zhen, R. Sutphen, W. Tan, Identification of Cell Membrane Protein Stress-Induced Phosphoprotein 1 as a Potential Ovarian Cancer Biomarker Using Aptamers Selected by Cell Systematic Evolution of Ligands by Exponential Enrichment, Anal. Chem., 86 (2014) 4521-4527. [25] A.M. Whited, Investigation of Impedance Spectroscopy for Detection of Ovarian Cancer, (2012). [26] W.S. Hummers, R.E. Offeman, Preparation of Graphitic Oxide, J. Am. Chem. Soc., 80 (1958) 1339-1339. [27] D. Li, M.B. Muller, S. Gilje, R.B. Kaner, G.G. Wallace, Processable aqueous dispersions of graphene nanosheets, Nat. Nanotechnol., 3 (2008) 101-105. [28] K. He, R. Liao, C. Cai, C. Liang, C. Liu, X. Chen, Y-shaped probe for convenient and label-free detection of microRNA-21 in vitro, Anal. Biochem., 499 (2016) 8-14. [29] F. Chen, C. Cai, X. Chen, C. Chen, “Click on the bidirectional switch”: the aptasensor for simultaneous detection of lysozyme and ATP with high sensitivity and high selectivity, Sci. Rep., 6 (2016) 18814. [30] H. Jang, Y.-K. Kim, H.-M. Kwon, W.-S. Yeo, D.-E. Kim, D.-H. Min, A
Graphene-Based Platform for the Assay of Duplex-DNA Unwinding by Helicase, Angew. Chem. Int. Ed., 122 (2010) 5839-5843. [31] H. Jang, S.-R. Ryoo, Y.-K. Kim, S. Yoon, H. Kim, S.W. Han, B.-S. Choi, D.-E. Kim, D.-H. Min, Discovery of Hepatitis C Virus NS3 Helicase Inhibitors by a Multiplexed, High-Throughput Helicase Activity Assay Based on Graphene Oxide, Angew. Chem. Int. Ed., 52 (2013) 2340-2344. [32]
M.J.
Solomon,
A.
Varshavsky,
Formaldehyde-mediated
DNA-protein
crosslinking: a probe for in vivo chromatin structures, P. Natl. Acad. Sci. USA, 82 (1985) 6470-6474. [33] J. Kennedy-Darling, L.M. Smith, Measuring the Formaldehyde Protein–DNA Cross-Link Reversal Rate, Anal. Chem., 86 (2014) 5678-5681. [34] J.D. McGhee, P.H. Von Hippel, Formaldehyde as a probe of DNA structure. I. Reaction with exocyclic amino groups of DNA bases, Biochemistry, 14 (1975) 1281-1296. [35] I. Taverniers, M. De Loose, E. Van Bockstaele, Trends in quality in the analytical laboratory. II. Analytical method validation and quality assurance, TrAC-Trend. Anal. Chem., 23 (2004) 535-552.
Biographies
Changqun Cai received her Ph.D in the college of chemistry from Xiangtan University in 2011, Xiangtan, Hunan, China. She holds the position of Associate Professor in Analytical Chemistry at College of Chemistry, Xiangtan University. Her research interests focus on Analytical Chemistry in Life Science and Biosensor. Hang Gong received his Ph.D in the college of chemistry from Xiangtan University in 2011, Xiangtan, Hunan, China. He worked as a faculty in College of Chemistry, Xiangtan University. His research interests focus on Analytical Chemistry in Life Science and organic synthetic methodology. Feng Chen is a M.S. student at Xiangtan University. Her research interests include spectral analysis and biosensor. Yi Liu is a M.S. student at Xiangtan University. Her research interests include spectral analysis and biosensor. Chunyan Chen received her Ph.D. in 2013 from Hunan University, Shangcha, Hunan, China. She worked as a faculty in College of Chemistry, Xiangtan University. She current research focus is chromatographic analysis. Xiaoming Chen is a professor of analytical chemistry in Xiangtan University, His research interests are chromatographic analysis and optical analysis.
Fig. 1. (A) AFM image and (B) height profile of GO, (C) FT-IR spectra of GO and RGO, (D) UV-Vis absorption spectra of GO and RGO in water
Fig. 2 Fluorescence spectra and variance of normalized fluorescence intensity with the concentration of STIP1: (1) 0, (2) 1, (3) 5, (4) 25, (5) 50, (6) 75 ng⋅mL-1. Excitation wavelength was 625 nm.
Fig. 3 Fluorescence spectra and variance of normalized fluorescence intensity with the concentration of CA125: (1) 0, (2) 0.05, (3) 0.1, (4) 0.5, (5) 1, (6) 2 U⋅mL-1. Excitation wavelength was 490 nm.
Fig. 4 Fluorescence responses of FAM-labeled CA125 aptamer (a), FAM-labeled CA125 aptamer + RGO (b), FAM-labeled CA125 aptamer + RGO + formaldehyde (c), FAM-labeled CA125 aptamer + RGO + formaldehyde + CA125 (d). Excitation wavelength was 490 nm. Inset: the DNA duplexes were analysed using agarose gel electrophoresis, (Lane1) FAM-labeled CA125 aptamer, (lane2) the mixture in FAM-labeled CA125 aptamer and formaldehyde.
Fig. 5 Study of the specificity of aptamers for the detection of CA125 and STIP1 (100 U⋅mL-1 for CA125; 1 ng⋅mL-1 for STIP1; 40 μg mL-1 for non-specific proteins).
A
B
Fig. 6 RLS spectra and the changed RLS intensity with the concentration of mixtures of CA125 and STIP1. (A) The changed RLS intensity at 419 nm. (B) The changed RLS intensity at 469 nm. Conditions: different concentrations of CA125: (1) 0, (2) 0.1, (3) 0.2, (4) 0.4, (5) 0.6, (6) 0.8, (7)1, (8) 2 U⋅mL-1; different concentrations of STIP1: (1) 0, (2) 1, (3) 2, (4) 4, (5) 8, (6) 10, (7) 20, (8) 40 ng⋅mL-1.
Scheme 1. Schematic representation of the assay of CA125 and STIP1 using aptamer-based fluorescent sensor (A) and RLS sensor (B).
Scheme 2 Schematic representation of the assay of CA125 via formaldehyde cross-linking.