In-situ Generation of Prussian Blue with Potassium Ferrocyanide to

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In-situ Generation of Prussian Blue with Potassium Ferrocyanide to Improve the Sensitivity of Chemiluminescence Immunoassay Using Magnetic Nanoparticles as Label Ning Yang, Yongxin Huang, Guosheng Ding, and Aiping Fan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01091 • Publication Date (Web): 13 Mar 2019 Downloaded from http://pubs.acs.org on March 13, 2019

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Analytical Chemistry

In-situ Generation of Prussian Blue with Potassium Ferrocyanide to Improve the Sensitivity of Chemiluminescence Immunoassay Using Magnetic Nanoparticles as Label Ning Yang, Yongxin Huang, Guosheng Ding, and Aiping Fan* School of Pharmaceutical Science and Technology, Tianjin Key Laboratory for Modern Drug Delivery & High Efficiency, and Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin 300072, PR China. Email address: [email protected] ABSTRACT: Using magnetic nanoparticles (MNPs) as a label in immunoassay (IA) possesses advantages such as high specific surface area, simple modification process. However, the catalytic activity of MNPs is low which limits their applications in IA. The present study found it interesting that potassium ferrocyanide reacts with MNPs leading to the in-situ generation of Prussian blue. The produced Prussian blue shows high catalytic activity on a luminol chemiluminescent (CL) reaction. Therefore, a simple and sensitive immunoassay for rIgG as model analyte using MNPs as label was developed. The CL intensity had liner increase with the concentration of rIgG ranged from 0.625 ng mL-1 to 20 ng mL-1. The limit of detection was calculated to be 0.59 ng mL-1. In addition, the applicability of this method was evaluated using the standard addition method. The recovery ranged from 80.0% to 115.0%. What’s more, the proposed CLIA method based on in-situ generation of Prussian blue with MNPs was also applied to the detection of carcinoembryonic antigen (CEA) and Hepatitis B virus (HBV)-related sequence-specific DNA. The LOD for the detection of CEA and sequence-specific DNA was estimated to be 0.28 ng mL-1 and 0.044 pmol, respectively.

Chemiluminescence immunoassay (CLIA) which couples the high sensitivity of chemiluminescence and good specificity of immunoassay has received great attention in recent years. The CLIA is widely applied in the field of clinical diagnosis and biological chemistry for the determination of tumor markers, cytokines, hormones, and so on.1-5 Currently, the natural enzymes such as horseradish peroxidase (HRP) which catalyze the oxidation of luminol by hydrogen peroxide were commonly used as labels for CLIA.6-7 Although the use of HRP is extensive, HRP, as a natural enzyme, is suffered from the high price, poor stability, complicated separation, and labeling processes, short shelf life, and so on. Hence, the development of mimetic peroxidase attracted great attention in recent years.8-10 In comparison with natural enzymes, nanomaterial mimics demonstrate advantages such as easy preparation, excellent stability, and so on. Many kinds of nanomaterial such as gold nanoparticles,11-12 silver nanoparticles,13 graphene oxide,14 carbon dots,15 gold nanoclusters,16 silver nanoclusters,17 Pd-Pt alloy nanodots,18 copper oxide,19 manganese dioxide,20 vanadium pentoxide,21 and so on were reported having mimetic peroxidase activity. In addition to catalyzing the oxidation of the chromogenic substrates such as 3, 3', 5, 5'tetramethylbenzidine (TMB) and 2, 2'-azino-bis (3ethylbenzothiazoline-6-sulfonic acid) (ABTS), many of them were reported having catalytic activity on the luminol-H2O2 CL reaction resulting in an enhanced CL signal. Many of them were employed in developing CL methods for the determination of hydrogen peroxide, glucose, uric acid, amino acids, and so on.22-26 In particular, some of them were employed as labels for the development of CLIA for the detection of tumor markers, human IgG, and cytokines.13, 27-30

MNPs possess advantages such as high specific surface area, good reaction affinity, and improved washing and separation efficiencies by using an external magnetic field. In previously, the MNPs were commonly employed as separation carries in the field of bioanalytical chemistry, biologic imaging, and drug delivery.31-34 In 2007, Yan group reported the mimetic peroxidase activity of Fe3O4 MNPs and applied the MNPs to an immunoassay for the detection of hepatitis B virus surface antigen and cardiac troponin I.35 Their work opened up the application of MNPs as a label in the immunoassay. In the present study, we found that the K4Fe(CN)6 was capable of improving the catalytic activity of Fe3O4 MNPs on the luminol CL reaction. Compared with the CL intensity of MNPscatalyzed luminol CL reaction, the CL signal enhanced 22 times with the introduction of K4Fe(CN)6 into the CL reaction. In 2010, Gu group prepared Prussian blue-modified -Fe2O3 MNPs and found that the peroxidase-like activity of the nanocomposite was improved with an increasing proportion of Prussian blue.36 Herein, although Fe3O4 MNPs instead of Fe2O3 MNPs was employed, we speculated that Prussian blue may be produced through the reaction of K4Fe(CN)6 and Fe3O4 MNPs and the enhanced CL signal may be attributed to the improved peroxidase-like activity of Prussian blue. Mechanism studies verified the above speculations. Based on the in-situ generation of Prussian blue with high catalytic activity, we established a simple and sensitive CLIA method by using Fe3O4 MNPs as a label in the present study.

Experimental section Chemicals and materials. Ultrapure water (18.24 MΩ cm-1) prepared through MILLI-XQ equipment was used throughout

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the whole work. 200 nm carboxyl-terminated Fe3O4 nanoparticles (COOH-MNPs) was purchased from Nanoeast Biotech. Co., Ltd. (Nanjing, China). N-(3dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) was purchased from J&K Scientific Ltd. (Beijing, China). Luminol was obtained from Alfa Aesar (Tianjin, China). K4Fe(CN)6, 2-(N-morpholine) ethane sulfonic acid (MES), and other chemical reagents were obtained from Guangfu Technology Development Co., Ltd. (Tianjin, China). Goat anti-rabbit IgG, biotinylated goat anti-rabbit IgG, and rabbit IgG were purchased from Boster Biological Technology Co. Ltd. (Wuhan, China). Streptavidin, human IgG, bovine serum albumin and bovine hemoglobin were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Rabbit serum was purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). The 96-well plates (Nunc™ MaxiSorp™) were purchased from Thermo scientific (Denmark). A stock solution of 0.1 M luminol was made by dissolving 0.177 g luminol in 0.1 M NaOH solution. The stock solution should be stored in the refrigerator (4 oC) for at least one week before use.14 10 mM phosphate buffer solution (PBS, pH 7.4) was prepared by dissolving 0.26 g KH2PO4, 2.9 g Na2HPO4·12 H2O, 8 g NaCl, and 0.2 g KCl in 1000 mL of ultrapure water. Washing buffer is 10 mM PBS (pH 7.4) containing 0.05% (w/v) Tween-20. 15 mM MES buffer solution (pH 5.5) was prepared by dissolving 0.29 g MES in 100 mL of ultrapure water and adjusting the pH to 5.5 with 0.1 M NaOH. Apparatuses. The CL detection was performed on Fluoroskan Ascent FL microplate reader with two dispensers (Thermo Scientific, USA). The separation of MNPs was completed by using a magnetic separator obtained from Shanghai Aorun Science and Technology Co., Ltd. The CL spectra were detected on a BPCL chemiluminescence analyzer (Beijing, China) with a series of high-energy optical filters of 320, 350, 380, 400, 440, 460, 490, and 535 nm.37 Preparation of streptavidin-modified MNPs (SA-MNPs). The SA-MNPs were prepared by conjugating streptavidin with COOH-MNPs through EDC reaction according to the method reported previously.38 In brief, 1 mL of COOH-MNPs (10 mg mL-1) was washed three times with 15 mM MES buffer (pH 5.5) and dispersed in 200 μL of MES buffer. Then, 500 μL of streptavidin (1 mg mL-1) was added and mixed with the COOH-MNPs. After incubating at 37 oC for 30 min, 250 μL of EDC (10 mg mL-1) was added in the mixture. After incubating for 2 hours at 37 oC, BSA as a blocking reagent was added into the solution and incubated or another 1 hour. Finally, the prepared SA-MNPs were washed three times with washing buffer and re-suspended in 1 mL of PBS containing 0.1% (w/v) BSA. The SA-MNPs were stored at 4 oC before use. The procedure of CL detection of K4Fe(CN)6-mediated luminol-MNPs CL reaction. In a typical experiment, 50 μL of 50 μM K4Fe(CN)6 was mixed with 50 μL of COOH-MNPs with a different concentration in the well of a 96-well plate and reacted for 40 min. The CL detection was performed on the Fluoroskan Ascent FL microplate reader. 100 μL of 100 μM luminol prepared by diluting a stock solution of luminol with Na2CO3-NaOH buffer solution (pH 12.0) was sprayed into the well of the 96-well plate through the dispenser of the Fluoroskan Ascent FL microplate reader, and the CL emission was detected at the same time. The procedure of CLIA for rIgG using SA-MNPs as a label. In a typical CLIA, the goat anti-rabbit IgG (1 mg mL-1)

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was diluted 1000-fold with coating buffer (0.1 M Na2CO3NaHCO3, pH 9.6). 100 μL of diluted goat anti-rabbit IgG was added into the well of a 96-well plate carefully and stored at 4 oC overnight. After washing with washing buffer for three times, 200 μL of 1% (w/v) BSA was added into the 96-well plate and incubated at 37 oC for 1 h to block the free site on the 96-well plate. After washing three times with washing buffer, 100 μL of different concentration of rIgG was added into the 96-well plate and incubated at 37 oC for 2 h. The wells were then washed three times with washing buffer. 100 μL of 1 µg mL-1 biotinylated goat anti-rabbit IgG was added into the well and incubated at 37 oC for 1 h. After washing three times with washing buffer, 100 μL of 100 μg mL-1 SA-MNPs was added into each well of the 96-well plate and incubated at 37 oC for 1 h. Finally, the 96-well plate was washed with washing buffer for three times and ultrapure water for one time respectively. For the CL detection, 50 μL ultrapure water and 50 μL of 50 μM K4Fe(CN)6 was added into the 96-well plated and reacted for 40 min. Finally, CL detection was performed as described above.

Results and Discussions Kinetic curves of K4Fe(CN)6-mediated luminol-MNPs CL reaction. We studied the kinetic curves of K4Fe(CN)6mediated luminol-MNPs CL reaction firstly (Figure 1). When 100 μM luminol was injected into 50 μM K4Fe(CN)6, a very weak CL signal was observed indicating that the luminol cannot be oxidized by K4Fe(CN)6 directly (curve c). An intense and flash type CL was produced if the K4Fe(CN)6 reacted with 10 μg mL-1 COOH-MNPs for 40 min beforehand, and the CL signal enhanced about 41-fold (curve a). Curve b is the CL kinetic curve of luminol-MNPs CL reaction which shows much lower CL intensity than that of luminol-MNPsK4Fe(CN)6 system. The above data suggested that K4Fe(CN)6 exhibited efficient enhancement effect on the luminol-MNPs CL reaction.

Figure 1. The kinetic curves of (a) MNPs-K4Fe(CN)6-luminol, (b) MNPs-luminol, and (c) K4Fe(CN)6-luminol, respectively.

Mechanism studies. As is known that K4Fe(CN)6 reacts with ferric ions (Fe3+) forming Prussian blue. In recently, Prussian blue was reported having mimetic peroxidase activity, and was capable of catalyzing the oxidation of 3,3',5,5'tetramethylbenzidine (TMB) by H2O2 producing a color change of TMB from colorless to blue.39 In addition, Gu group also reported that Prussian blue modified MNPs had higher catalytic activity than MNPs.36 Because Fe3+ ions also exist on the surface of MNPs, we assumed that the K4Fe(CN)6 may react with Fe3+ ions on the surface of MNPs producing


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Analytical Chemistry Prussian blue which can catalyze the oxidation of luminol by dissolved oxygen with high efficiency resulting in an enhanced CL signal. In order to identify the Prussian blue formed in the reaction of K4Fe(CN)6 and MNPs, the UV-Vis absorption spectrum and the FT-IR spectrum of the reaction products were recorded. As shown in Figure S1 (Supporting Information), the COOH-MNPs shows an absorption peak at 450 nm, and K4Fe(CN)6 has no absorption peak in the range of 400-800 nm. After reacting COOH-MNPs with K4Fe(CN)6 for 40 min, the final reaction mixture shows a new absorption peak located at 740 nm which is attributable to the formation of Prussian blue. Figure S2 (Supporting Information) shows the FT-IR spectrum of COOH-MNPs, K4Fe(CN)6, and their reaction products, respectively. The characteristic vibrations of K4Fe(CN)6 are the stretching vibration peak of CN- in Fe2+CN-Fe2+ at 2043 cm-1, and Fe2+-CN-Fe2+ at 417 cm-1. After reacting K4Fe(CN)6 with COOH-MNPs for 40 min, these two vibration peaks shift to 2089 cm-1 and 503 cm-1, respectively. They are assigned to the stretching vibration of CN- in Fe2+CN-Fe3+ and stretching vibration of Fe2+-CN-Fe3+, respectively. The above results suggested the generation of Prussian blue during the reaction of MNPs and K4Fe(CN)6. In order to confirm the existence and state of Prussian blue produced through the reaction of MNPs and K4Fe(CN)6, the TEM images of COOH-MNPs without and with the reaction of K4Fe(CN)6 was recorded. As shown in Figure S3B (Supporting Information), the square crystals which are Prussian blue were formed both in the solution and on the surface of MNPs. Secondly, we studied the CL spectrum of the K4Fe(CN)6 mediated luminol-MNPs CL reaction by using BPCL chemiluminescence analyzer with a series of high-energy optical filters of 320, 350, 380, 400, 440, 460, 490, and 535 nm. Figure S4 (Supporting Information) shows that the maximum CL emission wavelength is at 425 nm indicating the luminophore is still the excited-state 3-aminophthalate anions (3-APA*). How is the excited-state 3-APA* formed from luminol? According to previous reports,40-43 various reactive oxygen species are involved in the oxidation process of luminol. In order to investigate the mechanism, several kinds of free radical scavengers were added to the reaction solution. The results were shown in Table S2 (Supporting Information). Firstly, 87.4% CL intensity is inhibited with the addition of 10 U SOD which is a commonly used superoxide anion free radical (O2•-) scavenger. The above results suggest that the superoxide ions play an important role in the luminol CL reaction. The superoxide anion free radicals may be transformed from dissolved oxygen because the CL intensity declined by 69.1% when the dissolved oxygen was removed from the solutions by purging the reaction solutions with nitrogen for 40 min before the CL detection. The formed superoxide anion free radicals are unstable in solution, and may quickly convert into singlet oxygen (1O2) and H2O2. The role of 1O2 was verified by using singlet oxygen scavenger (histidine). 99.5% CL intensity was inhibited by histidine. In order to confirm the formation of H2O2 in the reaction, 18 U catalase which is capable of decomposing H2O2 was added in the CL reaction. 28.2% of quenching by the catalase suggested the existence of H2O2 in the CL reaction. The produced H2O2 may further react with ferrous ions leading to the formation of hydroxyl radical (•OH) based on Fenton reaction. The above hypothesis was verified by free radical quenching experiments. The CL intensity decreased about 40% with the addition of hydroxyl radical scavengers including methanol, ethanol, and

DMSO. Based on the above observation, the possible mechanism of the K4Fe(CN)6 mediated luminol-MNPs CL reaction is summarized in Scheme 1. The reaction of K4Fe(CN)6 and ferric ions on the surface of MNPs facilitated the formation of Prussian blue, which catalyzed the formation of reactive oxygen species including O2•-, 1O2, and •OH. The produced reactive oxygen species then react with luminol radical to form 3-APA*. When the 3-APA* returns to the ground state, a fast and intense CL signal is observed. Scheme 1. Possible mechanism of the K4Fe(CN)6-mediated luminol-MNPs CL reaction.

Optimization of experimental conditions. We assumed that the enhanced CL signal of K4Fe(CN)6-mediated luminolMNPs CL reaction may be derived from Prussian blue formed during the reaction of MNPs and K4Fe(CN)6. Hence, the experimental conditions which influence the formation of Prussian blue including the reaction time, the concentration of K4Fe(CN)6 and the acidity of reaction solution were investigated firstly (Figure 2). The reaction time of MNPs and K4Fe(CN)6 affects the amount of Prussian blue formed in the reaction. More Prussian blue would be formed with a longer reaction time. The CL intensity increased with the increase of reaction time ranging from 0 min to 40 min, then, maintained almost the same as the reaction time was longer than 40 min. In addition, the S/N ratio (refers to the ratio of CL intensity in the presence of MNPs to the CL intensity in the absence of MNPs) also reached a maximum when the reaction time was 40 min. Thus, 40 min of reaction time was employed for further studies. The concentration of K4Fe(CN)6 also plays an important role in the formation of Prussian blue. Higher concentration of K4Fe(CN)6 is a benefit to the formation of more Prussian blue leading to higher relative CL intensity (refers to CL intensity in the presence of MNPs minus CL intensity in the absence of MNPs). When the concentration of K4Fe(CN)6 increased from 0.1 µM to 50 µM, the relative CL intensity increased significantly indicating an increasing amount of Prussian blue was formed, and the CL intensity maintained almost the same as the concentration of K4Fe(CN)6 ranged from 50 µM to 100 µM. When the concentration of K4Fe(CN)6 was higher than 100 µM, the CL intensity decreased dramatically. We speculated that 50 µM K4Fe(CN)6 was enough for the transformation of ferric ions on the surface of MNPs to Prussian blue, and an excess amount of K4Fe(CN)6 may inhibit the CL signal because of the reducibility of K4Fe(CN)6. Hence, 50 µM K4Fe(CN)6 was selected for the following experiments.



Figure 2. Effects of (A) reaction time of MNPs and K4Fe(CN)6, (B) concentration of K4Fe(CN)6, (C) concentration of HCl on the CL reaction, (D) concentration of luminol on CL intensity. Experimental conditions for (A): COOH-MNPs, 10 μg mL-1; K4Fe(CN)6, 50 μM; HCl 5 mM; luminol, 100 μM; for (B), (C) and (D): reaction time, 40 min, other conditions were the same as (A).

The acidic environment is a benefit to the formation of Prussian blue.44 Thus, we investigated how the acidity of the reaction solution affected the CL signal by using different concentrations of HCl to adjust the acidity of the reaction solution. The CL intensity of the K4Fe(CN)6-mediated luminol-MNPs CL reaction increased monotonically with increasing concentration of HCl. However, strong blank signals were observed when the concentration of HCl was higher than 5 mM. Hence, 5 mM HCl was employed to control the acidity of the reaction of MNPs and K4Fe(CN)6. Luminol is the CL substrate in the K4Fe(CN)6-mediated CL reaction. Stronger CL intensity was observed with a higher concentration of luminol. The highest S/N ratio was obtained by using 100 µM luminol. Hence, 100 µM luminol was chosen for the following studies. CL response of COOH-MNPs and SA-MNPs. Under the optimal experimental conditions, the CL response of COOHMNPs was investigated. As shown in Figure 3, the CL intensity increased proportionally with increasing concentration of COOH-MNPs ranging from 0.046-0.75 μg mL-1. The lowest centration of COOH-MNPs (which is defined as the concentration of COOH-MNPs giving a CL intensity 3×the standard deviation of blanks) to trigger the CL reaction is estimated to be 0.0011 μg mL-1. In addition, we also tested the CL signal of SA-MNPs which will be used to construct immunoassay. The SA-MNPs were prepared by conjugating streptavidin with COOH-MNPs by using EDC reaction. The linear range for SA-MNPs was from 0.046-0.75 μg mL-1 with a detection limit of 0.0014 μg mL-1.

Figure 3. The CL intensity vs the concentration of COOH-MNPs and SA-MNPs.

Establishment of CLIA using SA-MNPs as a label. The present study developed a K4Fe(CN)6-mediated luminolMNPs CL reaction which showed much higher sensitivity than MNPs-catalyzed TMB colorimetric reaction, suggesting the feasibility of developing CLIA method with high sensitivity by using MNPs as a label. Hence, we applied the K4Fe(CN)6mediated luminol-MNPs CL reaction to the construction of CLIA for the detection of rabbit IgG (model analyte) using MNPs as label (Scheme 2). A classic sandwich type immunoassay was established by reacting different concentration of rIgG with primary antibody adsorbed on 96-


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Analytical Chemistry well plate and biotinylated secondary antibody, respectively. The sandwich type conjugates then reacted with SA-MNPs for the conjugation of MNPs on the surface of 96-well plated through the specificity reaction between biotin and streptavidin. After the immunoreaction, the CL signals were detected through the K4Fe(CN)6-mediated luminol-MNPs CL reaction. Scheme 2. The schematic illustration of the establishment of CLIA.

Figure 4A shows the CL responses with increasing concentration of rIgG. With increase concentration of rIgG in the sample the CL intensity increased significantly. A calibration graph in the range of 0.625-20 ng mL-1 showed a linear correlation (R2=0.9748) between the concentration of rIgG (ng mL-1) and the relative CL intensity, represented by I=0.0811C+0.333. The limit of detection was estimated to be 0.59 ng mL-1. Compared with colorimetric immunoassay using MNPs modified by Prussian blue prior to the immunoassay,36 our work performed much higher detection sensitivity for the target analyte (about 1000-fold). The parallel experiments have been done using identical immunoreagents and streptavidin-HRP conjugate. After the immunoreaction, HRPcatalyzed OPD colorimetric reaction was employed to detect the concentration of rIgG in the sample. The linear range for the detection of rIgG was 1.25-20 ng mL-1 with a detection limit of 0.9 ng mL-1 (Figure S5, Supporting Information). The CLIA method by using MNPs as the label was comparable with traditional ELISA using HRP as the label. The proposed CLIA using MNPs as the label possesses the following advantages. Firstly, the MNPs with various functional groups such as carboxyl-terminated, amino-terminated, azidoterminated, etc. are commercially available, which facilitates the conjugation of different kinds of biomolecules with MNPs. Secondly, MNPs can be magnetically separated by an external magnetic field. This property simplifies the purification process after the conjugation of biomolecules with MNPs. As for natural enzymes (such as HRP), the purification process is commonly tedious and time-consuming. Thirdly, MNPs have a large specific surface area which makes the possibility of modifying two (or more) kinds of biomolecules on the surface of MNPs, therefore, facilitates the coupling of further signal amplification method to improve the detection sensitivity. In addition, the reproducibility of the proposed CLIA was evaluated by a series of 7 repetitive measurements of 10 ng

mL-1 rIgG. The RSD was 6.8%, indicating the good reproducibility of the CLIA based on the K4Fe(CN)6-mediated luminol-MNPs CL reaction. In addition, the selectivity of the proposed method was evaluated by employing some interferents such as BSA, hemoglobin, human IgG, streptavidin, asparagine (Asp) and glycine (Gly) instead of rIgG as analytes in CLIA. As shown in Figure 4B, although more than the 10-fold concentration of interferents were employed in the CLIA, their CL signals were negligible demonstrating the good selectivity of the proposed CLIA method.

Figure 4. (A) The standard curve for the detection of rIgG by CL method using SA-MNPs as label (blank line) and ELISA method using HRP as label (red line); (B) the selectivity for the detection of rIgG. From a to g, followed by rIgG (10 ng mL-1); SA (100 ng mL-1); Human IgG (100 ng mL-1); Asp (10 nM); Gly (10 nM); BSA(1 mg mL-1); Heme (1 mg mL-1).

In order to evaluate the applicability of the proposed CLIA to real sample detection, human serum was employed to test the effect of complex matrix on the detection of target analyte. A standard addition method was employed herein. Three concentration levels of rIgG (5, 10, and 20 ng mL-1) were spiked in human serum which is diluted 50-fold with PBS. The concentration of rIgG was detected by using the proposed CL assay. The recovery of 5 ng mL-1, 10 ng mL-1 and 20 ng mL-1 rIgG was 115.0% ± 7.4%, 90.9% ± 20.5%, and 80.0% ± 0.9%, respectively. In addition, we detected the concentration of rIgG in a rabbit serum by using the proposed CLIA and ELISA, respectively. By using the traditional ELISA method, the concentration of rIgG was detected to be 1.51  0.37 mg mL-1. By using the proposed CLIA method, the concentration of rIgG was detected to be 1.43  0.14 mg mL-1, which is consistent with the result obtained from an ELISA method



using HRP as the label. The above results demonstrated that the proposed CLIA is capable of real sample detection. In order to evaluate the applicability of the proposed CL system for the detection of other biomolecules, we extended the in-situ generation of Prussian blue-based CL method for the detection of CEA and sequence-specific DNA related to HBV gene (The detailed material and procedures were shown in Supporting Information). The linear range for the detection of CEA was 0.5-100 ng mL-1 with LOD of 0.28 ng mL-1 (Figure 5A). In terms of the detection of sequence-specific DNA, the CL intensity increased significantly with the increasing amount of target DNA. A calibration graph in the range of 0.065-1.0 pmol showed a linear correlation (R2=9921) between the amount of target DNA (pmol) and the CL intensity, represented by I=5.4C+3.488. The detection limit was estimated to be 0.044 pmol (Figure 5B).

by using SA-MNPs as a label. The LOD in the rabbit IgG detection was 0.59 ng mL-1, which is comparable with the ELISA method coupled with HRP-catalyzed OPD colorimetric reaction. From the analytical chemistry point of view, the proposed CLIA can be easily extended to a large variety of clinical bioaffinity assays of analytes. The proposed CLIA method using MNPs as the label has the following advantages. Firstly, compared with traditional enzyme labels such as HRP, the MNPs is more stable, and the modified MNPs also exhibited good CL performance. Secondly, the MNPs provide not only a high specific surface area for the conjugation of biological recognition molecules but also high washing and separation efficiency during the labeling process by using an external easy-to-use magnetic field. Thirdly, the reaction of MNPs with K4Fe(CN)6 enhances the CL response significantly which improves the sensitivity of CLIA by using MNPs as a label. Finally, the preparation and surface modification techniques of MNPs are mature, and many kinds of MNPs with a different diameter or with different surface modification are commercially available which give a good prospect for application and dissemination of the proposed CLIA in the field of clinical diagnoses and biological assays.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Supplementary chemicals and materials. Procedure for the detection of CEA, sequence-specific DNA, and rIgG. The UV-Vis spectra, FT-IR spectra, and TEM images under different conditions. The CL spectra under different conditions. The standard curve of rIgG by using ELISA. The results of CL quenching experiments with various quenchers.

AUTHOR INFORMATION Corresponding Author *Fan Aiping: Email, [email protected]

Tel: +86-22-87440581. Fax: +86-22-87440581 Author Contributions The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.

Notes Figure 5. (A) CL response with increasing concentration of CEA. The inset is the linear relationship between the CL intensity and the logarithm value of CEA concentration. (B) The standard curve for CL detection of sequence-specific DNA by using MNPs as label.

Conclusion In summary, the present study developed a K4Fe(CN)6mediated luminol-MNPs CL reaction. Compared with the MNPs-catalyzed CL reaction, 22-fold enhanced CL signal was observed by reacting MNPs with K4Fe(CN)6 for 40 min in advance. The enhanced CL signal was attributed to the formation of Prussian blue through the reaction of MNPs and K4Fe(CN)6. Under the optimal experimental conditions, MNPs showed good CL response in the K4Fe(CN)6-mediated luminol-MNPs CL reaction. Based on the novel K4Fe(CN)6mediated luminol-MNPs CL system, a sandwich type CLIA was constructed for the detection of rabbit IgG (model analyte)

The authors declare no competing financial interest.

ACKNOWLEDGMENT We sincerely thank the financial support from the National Basic Research Program (973 program) of China “The fundamental and frontier studies of the new topology of molecular functional carbon materials” (No. 2015CB856500), National Natural Science Foundation of China (21475094), and Collaborative Innovation Center of Chemical Science and Engineering (Tianjin).

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In-situ Generation of Prussian Blue with Potassium Ferrocyanide to

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