A graphene-based chemiluminescence resonance energy transfer immunoassay for detection of phenothiazines in pig urine

Accepted Manuscript A graphene-based chemiluminescence resonance energy transfer immunoassay for detection of phenothiazines in pig urine

Ge Wang, Geng Nan Wang, Jian Ping Wang PII: DOI: Reference:

S0026-265X(19)30097-9 https://doi.org/10.1016/j.microc.2019.03.020 MICROC 3738

To appear in:

Microchemical Journal

Received date: Revised date: Accepted date:

12 January 2019 7 March 2019 7 March 2019

Please cite this article as: G. Wang, G.N. Wang and J.P. Wang, A graphene-based chemiluminescence resonance energy transfer immunoassay for detection of phenothiazines in pig urine, Microchemical Journal, https://doi.org/10.1016/ j.microc.2019.03.020

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ACCEPTED MANUSCRIPT A graphene-based chemiluminescence resonance energy transfer immunoassay for detection of phenothiazines in pig urine Ge Wang §, Geng Nan Wang §, Jian Ping Wang 1  College of Veterinary Medicine, Hebei Agricultural University, Baoding Hebei, China

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071000.

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Corresponding author. E-mail: [email protected] (Jian Ping Wang). The two authors contributed equally to this study. 1

ACCEPTED MANUSCRIPT Abstract In this study, a homogeneous competitive immunoassay based on graphene and chemiluminescence resonance energy transfer was first reported to detect small molecule substance. A generic monoclonal antibody of phenothiazine drugs was

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coupled to graphene as the recognition element and energy acceptor. The composite,

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the HRP-labeled hapten and the analyte solution were added into the microplate wells

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to perform homogeneous competition. Then luminol-H2O2-4-(imidazol-1-yl)phenol system was added to initiate light emission, and the light signal was positive

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correlation with the analyte concentration. After optimization of several parameters,

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the method was used to determine the residues of 4 phenothiazines in pig urine. Results showed that one assay was finished within 10 min, and the limits of detection

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for the 4 drugs were in the range of 2.0-5.0 pg/mL. Their recoveries from the

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standards fortified blank urine sample were in the range of 81.5%-96.8%. Therefore, this method could be used as a simple, rapid, and sensitive tool for routine screening

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the residues of phenothiazines in pig urine.

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Keywords: graphene; chemiluminescence resonance energy transfer; homogeneous immunoassay; phenothiazines; pig urine

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ACCEPTED MANUSCRIPT 1. INTRODUCITON Phenothiazines (PZs) are a class of sedatives that are usually used for the treatment of psychotic diseases in human beings. When PZs are used as veterinary drugs, they can cause calmness and reduce stress during the transportation of food producing

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animals [1]. However, the elimination of PZs from animal body will take a long time,

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and their residues in foods of animal origin can induce orthostatic hypotension and

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dermatological reactions to consumers [2,3]. For protection of consumer health, the European Union and China have forbidden the use of PZs in food producing animals

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[4,5]. Therefore, it is important to monitor their illicit uses. PZs are mainly excreted

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through urine [2,3], so the analysis of urine sample is a simple way to monitor their

abuses.

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By now, there have been many methods reported to determine PZs, such as liquid

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chromatography [6], mass spectrometry [7], chemiluminescence method [8], and enzyme linked immunosorbent assay (ELISA) [9-14]. Among these methods, ELISA is the most

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commonly used screening tool due to its high throughput analysis ability. However,

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ELISA has several limitations. Firstly, it usually requires several hours to coat and block the microplate. Secondly, it usually requires 1-4 hours to finish one assay. Thirdly, its result is based on colorimetric detection (optical density), so its sensitivity is only at ng/mL level. Fourthly, it requires the boring calculation to quantify the analyte. Chemiluminescence method (CL) is a rapid analytical method that can achieve much

higher sensitivity (pg/mL) than conventional ELISA. Therefore, CL has been

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ACCEPTED MANUSCRIPT introduced into ELISA to determine many analytes [15], such as chloramphenicol [16] and sulfamethoxypyridazine [17]. Results show that the method sensitivity is highly increased, but it still requires the coating, blocking and washing steps. Compared with the conventional ELISA and CL-ELISA, homogeneous immunoassay does not require

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the coating/blocking/washing steps, and the antibody-antigen binding is finished

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within several minutes, so the assay time can be largely shortened.

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Chemiluminescence resonance energy transfer (CRET) is a special homogeneous assay that involves the dipole-dipole energy transfer from a donor to an acceptor

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within close proximity (< 10 nm). Furthermore, CRET does not require an external

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light, so the non-specific signal is minimal. By now, CRET based methods have been used to determine various analytes [18]. For a CRET method, the selection of an

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appropriate energy acceptor is very important. Graphene is usually considered as the

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efficient acceptor among the numerous acceptors because it contains high planar surfaces and numberless energy-accepting sites. Therefore, graphene has been used to

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develop many CRET immunoassays for detection of various analytes [19-22].

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However, these CRET immunoassays are all used to detect biomolecules, and the assays are usually performed at sandwich-format. For immunoassay of small molecule substance, the assay should be performed at competitive format. To the best knowledge of the authors, there have been only several articles reporting homogeneous competitive immunoassays for detection of small molecule substances [23, 24]. In one report, the hapten-grafted graphene composite was combined with a fluorescence-labeled antibody to develop a

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ACCEPTED MANUSCRIPT fluorescence resonance energy transfer method for determination of bisphenol A [23]. In the other report, the sulfamethazine-functionalized quantum dots were combined with an enzyme-labeled antibody to develop a CRET method for determination of sulfamethazine [24]. As described above, a graphene-based CRET

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immunoassay for determination of small molecule substance has not been reported.

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immunoassay (GCRET-IA) for determination of PZs.

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Therefore, the objective of the present study was to develop a graphene-based CRET

In this study, a generic monoclonal antibody of PZs was coupled with graphene to

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produce a composite (G-Ab). The G-Ab, the analyte and a horseradish peroxidase

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(HRP)-labeled hapten were mixed to perform homogeneous competition, and then a highly effective luminol-H2O2-4-(imidazol-1-yl)phenol system was added to initiate

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light signal. As shown in Fig 1, the HRP-hapten bound with G-Ab when the analyte

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concentration was zero, so the proximity CRET occurred (no light signal). Conversely, the CRET was interfered when the analyte appeared, and the released HRP-hapten

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catalyzed the CL reagents to emit light, so the analyte amount was positive correlation

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with the CL intensity. Finally, the optimized method was used to determine PZs residues in pig urine, and the results were confirmed with an ultra performance liquid chromatography method (UPLC). 2. MATERIALS AND METHODS 2.1 Chemicals The standards of acepromazine (APZ), promethazine (PTZ), chlorpromazine (CPZ) and perphenazine (PPZ) were obtained from Dr. Ehrenstorfer (Ausburg, Germany).

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ACCEPTED MANUSCRIPT Horseradish peroxidase (HRP), 4-(imidazol-1-yl)phenol (IMP), p-iodophenol (IOP), 1-ethyl-3-(3’-dimethylaminopropyl)-carbodiimidehydrochlorde (EDC), and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Luminol was purchased from Acros (New Jersey, USA). Other

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chemicals were of analytical grade from Beijing Chemical Company (Beijing, China).

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Carboxyl graphene (5% of carboxyl group) and graphene oxide (6% of carboxyl

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group) were purchased from Nanjing Xfnano Materials Tech (Nanjing, China). The opaque 96-well microplates were purchased from Jingan Biotechnology Co., Ltd

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(Shanghai, China). The stock solutions of PZs standards (10.0 μg/mL) were prepared

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with methanol respectively, and their working solutions (0.0005-100 ng/mL) were obtained by diluting the stock solutions with phosphate buffer-saline (PBS, pH 7.2).

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PBS was prepared by dissolving 0.2 g KH2PO4, 0.2 g KCl, 1.15 g Na2HPO4, and 8.0

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g NaCl in 1000 mL water. The CL reagents (luminol, H2O2, IOP and IMP) were prepared with 0.1 M Tris-HCl (pH 8.6) respectively. The anti-chlorophenothiazine

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(anti-chPZ) monoclonal antibody and the haptens of phenothiazine and

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chlorophenothiazine were obtained in our previous studies [12,25]. 2.2 Preparation of G-Ab In this study, the antibody was immobilized on the surface of carboxyl graphene according to a previous report with minor modifications [20]. Briefly, 0.1 mg carboxyl graphene was suspended in 1 mL water, and 2 mg EDC and 4 mg NHS were added. The mixture was stirred gently in a 25 oC water bath for 30 min to activate the carboxyl groups. Then 1 mL PBS containing 1 mg Ab was added into the above

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ACCEPTED MANUSCRIPT suspension to be stirred gently at room temperature for 6 h. The suspension was washed with water and incubated in 2.0% BSA solution for 3 h to block the remaining activated sites on the surface of carboxyl graphene. The mixture was centrifuged at 15000 rpm at 4 oC for 30 min, and the supernatant was discarded. Finally, the

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obtained sediments were freeze-dried to obtain the composite G-Ab. The carboxyl

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graphene and the G-Ab were characterized by using Fourier transform infrared

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spectroscopy (FT-IR) and atomic force microscopy (AFM) respectively to verify the conjugation. For comparison, the antibody was also coupled with graphene oxide to

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prepare a controlled conjugate (GO-Ab) as the procedures described above.

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2.3 Preparation of HPR-haptens

The chlorophenotiazine hapten (chPZ) and phenotiazine hapten (PZ) were coupled

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to HRP respectively to prepare two competitive conjugates by using mixed anhydride

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method. Briefly, 3 mg hapten, 3 mL of DMF, 40 μL triethylamine, and 30 μL isobutyl chloroformate were added into a flask to be stirred gently at 4 oC for 1 h. Then the

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solution was added dropwise into 3 mL cold PBS containing 10 mg HRP (4 oC), and

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the mixture was stirred gently at 4 oC overnight. The solution was dialyzed in PBS for 3 days at 4 oC to obtain the conjugates (HRP-chPZ and HRP-PZ). HRP, the two haptens and the two conjugates were scanned on a UV spectrophotometer respectively to identify the conjugations. 2.4 Development of GCRET-IA The schematic representation of the GCRET-IA was shown in Fig 1, and the optimized procedures were performed as follows. Briefly, the G-Ab suspension was

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ACCEPTED MANUSCRIPT added into the wells of an opaque microplate (30 μL per well), and then 30 μL HRP-PZ and 30 μL analyte solution or pig urine were added into the wells. The plate was incubated at 37 oC for 7 min. Then, 30 μL luminol, 30 μL H2O2, and 30 μL IMP were added into the wells and the microplate was incubated at room temperature for 1

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min. Finally, the plate was put into Synergy HTX Multimode Reader (BioTek

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Instruments Inc., VT, USA) to record the CL intensity of each well. During the

H2O2 and IMP were optimized respectively.

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2.5 Method application

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experiments, the competition time and the concentrations of G-Ab, HRP-PZ, luminol,

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The pig urine sample was filtered with a 0.22 μm membrane for analysis without other preparation. About 1000 mL blank urine samples were obtained from several

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controlled pigs known to be free from PZs. The 4 PZs were fortified into the blank

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urine sample (0.1, 1, 10 ng/mL) to be analyzed respectively. The calibration curve was developed by plotting the analyte concentrations versus the CL intensities. The limit

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of detection (LOD) for each analyte was defined as the concentration corresponding

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to the mean signal of 20 blank urine samples plus three times of standard deviation. The intra-day recovery was the mean result of six repetitions in a single day, and the inter-day recovery was the mean result of duplicate analyses on six successive days. Finally, 30 real pig urine samples collected from several farms in China were analyzed with the GCRET-IA and confirmed with our recently reported UPLC method [26]. 3. RESULTS AND DISCUSSIONS

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ACCEPTED MANUSCRIPT 3.1 Characterization of G-Ab For development of a graphene-based CRET immunoassay, the anti-chPZ antibody was coupled with carboxyl graphene to prepare the composite G-Ab via the carboxyl groups. As shown in Fig 2A, the FT-IR results showed that carboxyl graphene only

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contained the peaks of carboxyl group (O-H 3435 cm-1, C=O 1627 cm-1, and C-O

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1392 cm-1), whereas G-Ab contained the peaks of carboxyl group (O-H 3356 cm-1,

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C=O 1757 cm-1) and the chemical groups from antibody (C-H 2897 cm-1, C=C 1597 cm-1, N-H 1243 cm-1 and C-N 1085 cm-1). This indicated that a new composite was

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obtained. Furthermore, the AFM results showed that the height of carboxyl graphene

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was 0.92 nm (Fig 2B), corresponding to monolayer graphene sheet. In contrast, G-Ab showed brighter spots with the height of 3.5 nm, and the difference was from the

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3.2 Evaluation of G-Ab

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antibody. This indicated that the G-Ab was obtained.

In order to verify if G-Ab could lead to CRET phenomenon, same amount of

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carboxyl graphene and G-Ab (10 μg/mL) were mixed with different concentrations of

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HRP-chPZ and pure HRP to perform the assay respectively. As shown in Fig 3, the CL intensities of graphene + HRP, graphene + HRP-chPZ and G-Ab + HRP increased rapidly as the enzyme concentration increased from 0.1 to 1.0 μg/mL. This meant that CRET phenomenon did not occur even though graphene was present in these mixtures. In comparison, the CL intensities of G-Ab + HRP-chPZ were negligible when the HRP-chPZ concentrations were in the range of 0.1-0.5 μg/mL, and the low CL intensities were obtained only when the HRP-chPZ concentrations were relatively

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ACCEPTED MANUSCRIPT excessive (0.8-1.0 μg/mL). This meant that G-Ab could induce CRET phenomenon. For comparison of quenching efficiency, different amounts of G-Ab and GO-Ab were mixed with HRP-chPZ (1 μg/mL) to perform the assay respectively. As shown in Fig 4, the CL intensities decreased rapidly when the G-Ab concentration increased

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from 0 to 20 μg/mL, and the quenching degree was about 99%. In contrast, the CL

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intensities decreased slowly when using GO-Ab (0-20 μg/mL), and the quenching

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degree was about 56%. These results indicated that graphene was a more suitable energy acceptor for CRET method than GO, which was similar to the previous report

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[20]. Therefore, G-Ab was selected for the subsequent experiments.

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3.3 Characterization of two HRP-labeled haptens

For a competitive immunoassay, an enzyme labeled hapten was required. In this

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study, chPZ and PZ were coupled to HRP to prepare two conjugates respectively. As

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shown in Fig 5A, the representative UV diagram of HRP-PZ contained the characteristic peaks of HRP and PZ, indicating the successful conjugation. During the

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experiments, HRP-PZ and HRP-chPZ were mixed with G-Ab and series

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concentrations of CPZ solutions (0.01-10 ng/mL) to perform the assay respectively. As shown in Fig 5B, the CL intensities when using HRP-PZ were higher than that when using HRP-chPZ. This was because the antibody was generated against chPZ, so HRP-chPZ showed higher binding ability to the antibody than HRP-PZ. When HRP-chPZ was mixed with CPZ and G-Ab, more HRP-chPZ molecules bound with G-Ab to induce CRET, thus obtaining low CL intensity. In other words, the use of HRP-PZ could obtain higher CL intensity and higher sensitivity than the use of

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ACCEPTED MANUSCRIPT HRP-chPZ. Therefore, HRP-PZ was selected for the subsequent experiments. 3.4 Optimization of GCRET-IA For the GCRET-IA, the concentrations of G-Ab and HRP-PZ were two critical factors because their concentrations directly influenced the CL intensity and method

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sensitivity. During the experiments, different concentrations G-Ab and HRP-PZ were

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used to perform the assay with CPZ as the test drug. As shown in Fig 6, the CL

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intensity reached a plateau when using 10 μg/mL G-Ab and 0.8 μg/mL HRP-PZ, so the two parameters were selected for the subsequent experiments. For shortening the

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assay time, G-Ab, HRP-PZ and CPZ solution were added into the microplate wells for

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incubation for different times (5-60 min). Results showed that the CL intensity reached a plateau when the mixture was incubated for 7 min, so 7 min was selected as

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the optimal competition time.

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HRP catalyzed luminol-H2O2 system is the most popular CL system [15], and the addition of an enhancer into this system can highly increase the light signal [27-29].

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In this study, the analyte amount was positive correlation with the CL intensity, so the

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concentrations of the used CL reagents were optimized. As shown in Fig 7, the CL intensity of the test drug CPZ reached the highest when using 6 mmol/L luminol and 8 mmol/L H2O2, so the two parameters were selected for the subsequent experiments. Furthermore, the CL intensities of the test drug CPZ with and without an enhancer were compared (luminol-H2O2, luminol-H2O2-IOP, and luminol-H2O2-IMP). As shown in Fig 8, the maximum CL intensities when adding IMP and IOP were much higher than that without an enhancer. In comparison, the use of IMP achieved higher

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ACCEPTED MANUSCRIPT CL intensity than the use of IOP, which was consistent with the previous reports [27,28]. Therefore, 1 mmol/L IMP was selected for the subsequent experiments. 3.5 Determination parameters of GCRET-IA Under the optimal conditions, the four PZs were diluted with the blank pig urine to

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perform the assay respectively. As shown in Table 1, the linearity ranges for the 4

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analytes were in the range of 0.01-20 ng/mL, the correlation coefficients (r2) were in

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the range of 0.9974-0.9992, and the LODs were in the range of 2.0-5.0 pg/mL. As shown in Fig. 9, the representative calibration curve of urine-matrix matched CPZ

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was similar to that of CPZ standard, illustrating the minimum matrix effect. For

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evaluation of method selectivity, four other sedatives were also diluted with the blank urine for analysis respectively (diazepam, haloperidol, azaperone, estazolam, 0.01-20

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ng/mL). Results showed that their CL intensities were generally comparable to the

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blank urine sample, indicating the method was only specific for PZs. The representative curve for diazepam was shown in Fig. 9.

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3.6 Method application

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The 4 PZs were fortified into the blank pig urine at three levels for analysis respectively. As shown in Table 1, the intra-assay recoveries were in the range of 81.5%-96.8%, the inter-assay recoveries were in the range of 84.7%-94.5%, and the coefficients of variation (CV) were in the range of 5.1%-10.2%. Finally, the 30 real pig urine samples were analyzed with the immunoassay, and the results were confirmed with the UPLC. Results showed that two real urine samples were determined as positive by the immunoassay (1.2 and 16.5 ng/mL, calculated as CPZ

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ACCEPTED MANUSCRIPT because the antibody could not distinguish the 4 PZs), and other samples were determined as negative. The UPLC confirmation showed that the two urine samples contained CPZ (1.7 and 20.3 ng/mL), and other real urine samples were all negative. This meant that the GCRET-IA method could be used for rapid screening the residues

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of the 4 PZs, but the positive samples should be confirmed with an instrumental

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method to verify the specific analyte.

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3.7 Comparison with the related methods

The previous graphene-based CRET immunoassays were all used to detect

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macromolecules, and the assays were all performed at sandwich format [19-22]. The

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only one graphene-based homogeneous immunoassay for small molecule substance was based on fluorescence resonance energy transfer [23]. This study for the first time

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reported a GCRET-IA method for determination of small molecule substance. For

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comparison, the details of previously reported ELISA methods for PZs are summarized in Table 2. As shown in Table 2, these ELISA methods were performed at

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indirect competitive format, required the coating/blocking/washing/incubating steps

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(12-17 h of assay time), and obtained low sensitivity (with LODs of 0.03-15.3 ng/mL) [9-14]. General comparison of assay procedure, sensitivity and time-consumption, the GCRET-IA method showed better performances than the previous immunoassays. 4. CONCLUSION This study for the first time developed a graphene based CRET homogeneous immunoassay for determination of small molecule substance (PZs). This method did not require the tedious coating, blocking and washing steps, and the total time of

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ACCEPTED MANUSCRIPT finishing one assay was about 10 min, so it was much rapider and simpler than the conventional ELISA method. Furthermore, the method achieved ultrahigh sensitivity. Therefore, the GCRET-IA could be used as a rapid, simple, and ultra-sensitive method for routine screening the residues of PZs in pig urine.

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ACKNOWLEDGEMENTS

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This study was financed by National Natural Sience Foundation of China (No.

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31271869), and Shijiazhuang Technology Incubation Project (171550089A). Compliance with Ethical Standards

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All of the authors declare that they have no conflict of interest.

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Graham ex Benth, Luminescence. 30 (2015) 568-575.

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ACCEPTED MANUSCRIPT Figure captions: Fig 1. The schematic representation of the GCRET-IA. Fig 2. (A) FT-IR and (B) AFM results of (a) carboxyl graphene and (b) G-Ab. Fig 3. Mean CL intensities of the mixtures of carboxyl graphene and G-Ab (10 μg/mL)

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with different concentrations of HPR and HRP-chPZ (luminol 5 mmol/L; H2O2 5

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mmol/L; n=6).

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Fig 4. Mean CL intensities of the mixtures of HRP-chPZ (1.0 μg/mL) with different concentrations of G-Ab and GO-Ab (0, 1, 2, 5, 8, 10, 15 and 20 μg/mL) (luminol 5

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mmol/L; H2O2 5 mmol/L; n=6).

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Fig 5. (A) UV scanning diagrams of PZ, HRP and HRP-PZ. (B) Mean CL intensities for detection of CPZ when using two competitive conjugates (CPZ, 0.01-10 ng/mL;

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H2O2 5 mmol/L; n=6).

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enzyme amount 1.0 μg/mL; G-Ab, 10 μg/mL; competition 30 min; luminol 5 mmol/L;

Fig 6. Mean CL intensities for detection of CPZ when using different concentrations

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of G-Ab and HRP-PZ (CPZ 1.0 ng/mL; competition 30 min; luminol 5 mmol/L; H2O2

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5 mmol/L; n=6).

Fig 7. Mean CL intensities for detection of CPZ when using different concentrations of luminol and H2O2 (G-Ab 10 μg/mL; HRP-PZ 0.8 μg/mL; competition 7 min; CPZ 1.0 ng/mL; n=6). Fig 8. Mean CL intensities for detection of CPZ when using different enhancers (G-Ab 10 μg/mL; HRP-PZ 0.8 μg/mL; CPZ 1.0 ng/mL; competition 7 min; luminol 6 mmol/L; H2O2 8 mmol/L; n=6).

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ACCEPTED MANUSCRIPT Fig 9. Calibration curve of urine-matrix matched CPZ and standard CPZ (analyte 0.01-20 ng mL-1; G-Ab 10 μg/mL; HRP-PZ 0.8 μg/mL; competition 7 min; luminol 6

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mmol/L; H2O2 8 mmol/L; IMP 1 mmol/L; n=6).

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ACCEPTED MANUSCRIPT Table 1 Recoveries of the 4 PZs from the fortified blank pig urine (n=6) Inter-assay

0.01-20

0.9985

2.0

PTZ

0.02-20

0.9974

3.0

PPZ

0.02-20

0.9991

5.0

CPZ

0.01-20

0.9992

2.0

0.1 1 10 0.1 1 10 0.1 1 10 0.1 1 10

Intra-assay

Recovery (%)

CV (%)

Recovery (%)

CV (%)

85.2 88.9 91.5 93.1 84.7 86.3 87.5 93.5 92.4 94.0 93.2 94.5

10.2 7.4 6.3 7.4 5.9 5.1 8.9 5.3 6.3 6.4 7.4 6.0

84.6 81.5 83.6 88.9 94.5 95.3 92.1 87.2 96.8 90.5 93.4 92.6

7.8 5.3 5.6 6.8 7.3 5.6 9.5 6.4 5.8 9.9 6.7 7.2

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APZ

Added (ng/mL)

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r

LOD (pg/mL)

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2

AC

CE

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D

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Analyte

Linearity (ng/mL)

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ACCEPTED MANUSCRIPT Table 2 Comparison with the previous ELISA methods for PZs total time (coating+blocking+assay)

sample

reference

CPZ

0.31-0.46

overnight+1 h+4 h

animal tissues

9

CPZ

0.03

overnight+3 h+2 h

animal tissues

10

0.2-0.4

overnight+0.5 h+2 h

swine tissues

11

0.5-0.8

overnight+0.5 h+2 h

meat

12

5 PZs

0.1-1.8

overnight+0.5 h+2 h

animal tissues

13

9 PZs

1.1-15.3

overnight+0.5 h+1 h

feeds

14

0.002-0.005

10 min

urine

This study

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D

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competitive homogeneous immunoassay

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4 PZs

CE

3 PZs

indirect competitive ELISA

AC

5 PZs

method

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LOD (ng/mL)

analyte

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ACCEPTED MANUSCRIPT Fig 1 CRET

+ sample

luminol/H2O2 No light = Negative Step B

HRP-labeled hapten

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PT

Step A

analyte

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graphene-labeled antibody

AC

CE

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D

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Light = Positive

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ACCEPTED MANUSCRIPT Fig 2

b

CE

PT E

D

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a

AC

B

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RI

PT

A

25

ACCEPTED MANUSCRIPT Fig 3

220000 200000 180000 160000

HRP 0.1g/mL HRP 0.2g/mL HRP 0.5g/mL HRP 0.8g/mL HRP 1.0 g/mL HRP-chPZ 0.1g/mL HRP-chPZ 0.2g/mL HRP-chPZ 0.5g/mL HRP-chPZ 0.8g/mL HRP-chPZ 1.0g/mL

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120000 100000 80000

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CL intensity

140000

40000 20000 0

mixed with G-Ab

AC

CE

PT E

D

MA

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mixed with graphene

SC

60000

26

ACCEPTED MANUSCRIPT Fig 4

G-Ab (quench degree of 0-99%) GO-Ab (quench degree of 0-56%)

160000

80000

PT

CL intensity

120000

SC

RI

40000

0 0

5

10

15

20

AC

CE

PT E

D

MA

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Concentration of G-Ab or GO-Ab (g/mL)

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ACCEPTED MANUSCRIPT Fig 5

B

160000

D

HRP-PZ HRP-chPZ

0

PT E CE

80000

0

AC

CL intensity

120000

40000

MA

NU

SC

RI

PT

A

2

4

6

8

10

CPZ concentration (ng/mL)

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ACCEPTED MANUSCRIPT Fig 6

20000

PT

10000

b (g

15

20

0.2

m

AC

CE

PT E

D

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/mL)

10

( g/

8

G-A

0.6 0.4

PZ

5

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2

1.0 0.8

P-

0

HR

SC

5000

L)

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CL intensity

15000

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ACCEPTED MANUSCRIPT Fig 7

25000

PT

15000

RI

10000

4

l/L)

2

2

) /L ol m (m

6

mmo

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nol (

AC

CE

PT E

D

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lumi

6 4 2

8

O

10

10

8

2

0

SC

5000

H

CL intensity

20000

30

ACCEPTED MANUSCRIPT Fig 8

38000

without enhancer IOP IMP

36000

32000

PT

CL intensity

34000

30000

RI

28000

24000 0

1

2

3

SC

26000

4

5

6

AC

CE

PT E

D

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Enhancer concentration (mmol/L)

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ACCEPTED MANUSCRIPT Fig 9 350000

300000

Urine matched CPZ

y = 16060x + 3360.4 R 2 = 0.9981

Standard CPZ

y = 14792x + 6144.2 R 2 = 0.9965

Urine matched diazepam

200000

150000

PT

CL intensity

250000

RI

100000

0 0

5

10

SC

50000

15

20

AC

CE

PT E

D

MA

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Concentration (ng/mL)

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ACCEPTED MANUSCRIPT

Highlights 1. An antibody was coupled with graphene to synthesize a novel composite

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2. A competitive CRET immunoassay was developed for detection of phenothiazines

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3. One assay including all experiment steps was finished within 10 min

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4. The method achieved ultrahigh sensitivity with limit of detection 2-5 pg mL-1

33