Technical Note pubs.acs.org/ac

Electrogenerated Chemiluminescence Bioanalytic System Based on Biocleavage of Probes and Homogeneous Detection Jing Zhang, Honglan Qi,* Zhejian Li, Ni Zhang, Qiang Gao, and Chengxiao Zhang* Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an, 710062, People’s Republic of China S Supporting Information *

ABSTRACT: A novel electrogenerated chemiluminescence (ECL) bioanalytic system based on biocleavage of a ECL probe and homogeneous detection was designed and utilized for the first time for highly sensitive quantification of proteases to overcome drawbacks from probes directly immobilized on electrodes and commercial ECL biosystems, based on bioaffinity reactions. Prostate-specific antigen (PSA) was taken as a model analyte and ruthenium complex-tagged specific peptide (CHSSKLQK) was designed as an ECL probe (peptide-Ru1). ECL bioconjugated magnetic beads were synthesized through a simple solid-phase synthesis. When analyte PSA was introduced into the suspension of ECL bioconjugated magnetic beads, a biocleavage of the peptide occurred and the cleaved Ru1 part was released from the magnetic beads. ECL measurement was carried out in the presence of co-reactant tripropylamine, using two models. One is homogeneous ECL detection on a bare graphite pencil electrode (PGE), and the other is enriching ECL detection after the cleaved Ru1 part of the peptide was concentrated into the surface film of Nafion/gold nanoparticles modified PGE (AuNPs/Nafion/PGE). The extremely low detection limit of 80 fg/mL and high reproducibility (relative standard deviation (RSD) of 5.4% for six measurements of 0.5 pg/mL) for the detection of PSA were achieved at AuNPs/Nafion/PGE. This work demonstrates that the bioanalytic system designed can not only quantify proteases with high sensitivity and selectivity, but also diminish the complicated electrode process and improve the reproducibility by conducting the biocleavage and transduction steps at different surfaces. It can be easily extended for ECL analysis of other proteases in this system and other detection techniques, including optics and electrochemistry.

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homogeneous detection incorporating magnetic deposition have not been reported. Therefore, in this work, we describe the design and development of a bioanalytic system based on the biocleavage of probes and homogeneous detection for highly sensitive quantifying proteases to overcome drawbacks from probes directly immobilized on electrodes and commercial ECL biosystems based on bioaffinity reactions, and to improve sensitivity for the detection of protease. Protease is any enzyme that performs proteolysis, that is, begins protein catabolism via the hydrolysis of the peptide bonds. It is closely related to a large number of vital processes in the body’s metabolic processes, including the cell growth, cell death, tissue remodeling, and immune defense, and it is also used as biomarkers.7 Since many proteases express low abundance in biological systems, it is of great significance in life science research to develop highly sensitive approaches for the detection of proteases.8 Bioanalytic sensing systems are frequently used to assay the proteases, based on biocleavage of

esign and development of novel bioanalytic systems for quantifying proteins, genes, and bacteria are of great importance in fundamental research and clinical tests, and drug screening.1 From an analytical chemistry viewpoint, there are mainly two approaches: separation-based HPLC/CE/MS and biological molecular recognition-based biosensors/biosensing. Separation-based HPLC/CE/MS systems are powerful, because they can conduct identification and quantification of several thousands components.2 For the quantification of known components, biosensors/biosensing systems are easily performed with low-cost instruments in central Lab.3 Electrogenerated chemiluminescence (ECL) biosensors/biosensing systems have been largely accelerated by the use of commercially ECL instruments, such as Elecsys.4 These ECL detection systems are adapted to measure ECL labels present on the surface of magnetically responsive beads in the presence of tripropylamine. Most of the bioanalytical systems were mainly performed on the basis of signal changes after a bioaffinity reaction between the ECL probes (biological molecular recognition elements labeled with ECL labels) and target analytes.5,6 This system was widely used in clinic tests for more than one hundred items in clinical tests. However, ECL bioanalytical systems based on biocleavage of probes and © XXXX American Chemical Society

Received: April 7, 2015 Accepted: June 1, 2015

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DOI: 10.1021/acs.analchem.5b01396 Anal. Chem. XXXX, XXX, XXX−XXX

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Figure 1. Schematic representation of ECL bioanalytic system based on the biocleavage of ECL bioconjugated magnetic beads and ECL detection on PGE and AuNPs/Nafion/PGE.

2. EXPERIMENTAL SECTION The materials, apparatus, and synthesis of the ECL bioconjugated magnetic beads (Fe3O4@Au-peptide-Ru1) are presented in the Supporting Information. AuNPs with a diameter of ∼13 nm (Figure S-2 in the Supporting Information) were prepared by the reduction of HAuCl4 with citrate in aqueous solution following the literature method.24 The concentration of AuNPs was calculated to be 9.55 nM (ε = 2.08 × 108 L (mol cm)−1 at 524 nm).25,26 AuNPs/Nafion/PGE was fabricated by simply spin-coating 10 μL of AuNPs−Nafion mixture solution, according to our previous report.15 Two detection models were employed for ECL measurements, as shown in Figure 1. Ten microliters (10 μL) of ECL bioconjugated magnetic beads (1.6 mg/mL) was added into a 1 mL plastic tube containing 90 μL of PSA solution or serum samples, to perform a cleavage reaction for 40 min at 30 °C and then to force the beads attach to the side wall of the tube under a perpetual magnetic separator for 5 min. For homogeneous ECL detection on a bare PGE, a PGE was immersed in the mixed solution that 100 μL of 0.10 M phosphate buffered saline (PBS, pH 7.4) containing 100 mM TPA was mixed with 100 μL magnetic deposition supernatant. For enriching ECL detection, AuNPs/Nafion/PGE was immersed in 100 μL of the cleaved solution under magnetic deposition to accumulate the cleaved Ru1 part of the peptide for 25 min, and was washed with 10 mM PBS. The accumulated electrode then was transferred into a ECL cell containing 0.10 M PBS (pH 7.4) and 50 mM TPA. ECL measurement was performed using a linear sweep with a scan rate of 50 mV/s. The concentration of PSA was quantified by an increased ECL intensity,

the probes, which consist of a specific substrate peptide and label, such as radioactive,9 fluorescent,10−12 chemiluminescence,13 electrochemical,14 and ECL reagents.15 There are two approaches that include biosensors and biosensing systems, for quantifying proteases. In the first approach, the biosensor was fabricated by directly immobilizing the probes (electroactive,16,17 ECL reagent18 labeled peptide substrates) on the surface of electrodes. When the biosensor is incubated with the target protease, the signal is directly related to the amount of uncleavage of the probes on the electrode surface. This signaloff approach displays a high sensitivity and speediness. However, its detection limit is not very low and the reproducibility is low, because of the fouling of the electrodes and one shot. In the second approach, the probe is linked to magnetic beads/solid surface.19 When the probe-immobilized beads are incubated with the target protease, the signal is directly related to the concentration of cleavage of the probes in the test solution. This signal-on approach displays high sensitivity, selectivity, and reproducibility since separation of cleavage step and measurement step. In addition, a small portion of the probe magnetic beads prepared is only used in each test, so that they can be used many times. However, this approach commonly accompanied a solution dilution when the volume of testing solution is larger than that of sample solution. In this work, for the first time, we proposed a new strategy that employs ECL bioconjugated magnetic beads as probes and enriches the cleavage probes onto graphite pencil electrode (PGE) for the ECL detection of protease. Prostate-specific antigen (PSA), which is a 30 kDa kallikrein-like serine protease, is chosen as an analyte, since it is the most reliable biomarker for early diagnostics of prostate cancer and monitoring the recurrence of the disease. 2 0−2 2 A specific peptide (CHSSKLQK; see Figure S-1 in the Supporting Information) is designed, according to Denmeade et al.,23 as a substrate. Figure 1 shows the schematic representation of ECL bioanalytic system. The ECL detection was performed after the enzyme cleavage step and the enriching step. In this paper, the synthesis and characterization of the ECL bioconjugated magnetic beads and the characterization of AuNPs/Nafion/PGE are discussed, and the analytical performance for the determination of PSA is presented.

ΔI = Is − I0

where I0 is the blank signal and IS is the ECL intensity after being incubated with PSA.

3. RESULTS AND DISCUSSION 3.1. ECL Bioconjugated Magnetic Beads. It is reported that the silica magnetic nanoparticles as a carrier show higher biocompatibility and higher ECL stability.27 However, the substrate peptide is required to couple the surface of silica magnetic nanoparticles covalently. In this work, Fe3O4@Au B

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Figure 2. (A) Two-step immobilization of peptide and Ru1 on Fe3O4@Au. (B) TEM image of Fe3O4@Au (inset shows a high-resolution transmission electron microscopy (HRTEM) image of Fe3O4@Au). (C) Fluorescence CCD imaging of Fe3O4@Au-peptide-Ru1. (D) ECL intensity versus potential curves at Fe3O4@Au-peptide-Ru1-modified GCE in 0.10 M PBS (pH 7.4) (a) with and (b) without 50 mM TPA. Scan rate = 50 mV/s.

Fluorescence experiments demonstrated that the Ru1 is coupled with the peptide on the surface of the magnetic beads and the ECL probe on the magnetic beads can be cleaved by the target PSA (see Figure S-3 in the Supporting Information). The fluorescence spectra in the supernatant of Fe3O4@Aupeptide-Ru1 with 100 ng/mL PSA is similar to that of Ru1 in solution. This is attributed to the same fluorescence compound Ru1 in both solutions. The fluorescence intensity of the supernatant of Fe3O4@Au-peptide-Ru1 under a magnetic field, in the presence of 100 ng/mL PSA, is much higher than that in the absence of PSA and that in the presence of BSA. This is attributed to the fact that PSA can only cleave the peptide, which allows Ru1 to enter the supernatant. The results indicate that the ECL bioconjugated magnetic beads is successfully prepared for the potential detection of target PSA. Furthermore, the storage stability of the ECL bioconjugated magnetic beads was checked by observing the precipitation by exposing them to water and 0.1 M PBS. It was found that no precipitation occurs at least one month (see Figure S-4 in the Supporting Information). This indicates that the prepared beads have long-term storage stability in different aqueous media. All of the above experimental results indicate that the methodology that has been proposed can be used to prepare the ECL bioconjugated magnetic beads successfully. 3.2. Detection Electrodes. In the proposed biosensing system, the detection electrode used is one of key issues involving the sensitivity and selectivity and speediness. Bare electrode (GCE, PGE) benefits simplicity and speediness. Nanomaterial-modified electrodes benefit the sensitivity and selectivity. Considering that the signal compound (the cleaved Ru1 part of the peptide) is dissolved into the solution, which led to diluting the concentration of the signal compound in solution, we designed Nafion film and AuNPs on the detection electrode to concentrate the signal compound and to enhance the ECL intensity. In this work, we explored both of the electrodes for the detection of PSA. 3.2.1. Bare Electrodes. PGE was, for the first time, chosen as a base electrode in this work, because it has high electrochemical reactivity and low cost, and it can be easily modified

magnetic beads is chosen as a carrier, because the peptide is easy to be self-assembled on the surface of Fe3O4@Au magnetic beads through the Au−S bond and the prepared ECL bioconjugated magnetic beads is one-shot. There are two ways to prepare the ECL bioconjugated magnetic beads. The first is performed by self-assembling the synthesized ECL probe (bis(2,2′-bipyridine)-4′-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester labeled peptide, Ru1-peptide) onto the surface of Fe3O4@Au magnetic beads via cysteine. The separation of the ECL probe is required to use Sephedex 15 column or dialysis.28 The second is done by self-assembling the peptide onto the surface of the beads and then covalently coupling Ru1 with the self-assembled peptide. This is simpler than the first way, because only magnetic separation is used. Therefore, the second way was used, as shown in Figure 2A. Figure 2B shows the TEM image of the Fe3O4@Au. The average diameter of the Fe3O4@Au is 40−50 nm, and their distribution is well-scattered. Zeta potential of the Fe3O4@Au dispersed in water had a negative potential of −22 mV, indicating that the Fe3O4@Au has a negatively charged surface.29 After the peptide was self-assembled and Ru1 was coupled onto the surface of Fe3O4@Au, the zeta potential was reversed to be positive (+11 mV). This change is attributed to the fact that the positively charged Ru1 was covalently coupled with the peptide on the surface of the magnetic beads. This suggests that the peptide and Ru1 are coupled on the surface of Fe3O4@Au. Figure 2 C shows the fluorescence imaging of the ECL bioconjugated magnetic beads. The emission, attributed to the Ru1 emission at 627 nm, indicates that both the Ru1 and the peptide are coupled on the surface of Fe3O4@Au. Figure 2D shows ECL intensity versus potential curves at an Fe3O4@ Au-peptide-Ru1-modified GCE fabricated by drop-coating 10 μL of 1.6 mg/mL Fe3O4@Au-peptide-Ru1 onto the surface of GCE. One obvious ECL peak at 1.17 V appeared at Fe3O4@ Au-peptide-Ru1-modified GCE in the presence of TPA, which is ascribed to the radical TPA• (formed during TPA oxidation), which reduces Ru(bpy)33+ (formed from the oxidation of Ru(bpy)32+) to produce Ru(bpy)32+*.30,31 This indicates that the prepared beads have the expected ECL properties. C

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Figure 3. ECL intensity versus potential curves for 20 pg/mL PSA using (a,d) bare, (b,e) Nafion, and (c,f) AuNPs/Nafion modified electrodes (panels a, b, and c show data for GCE transducers and panels d, e, f for PGE transducers. Scan rate = 50 mV/s; 25 min of preconcentration time at the Nafion-modified electrode.

and renewed.32,33 In order to illustrate the good performance of PGE, ECL performance of Ru1 at GCE was compared with that at PGE (see Figure S-5 in the Supporting Information). The ECL intensity versus potential curves showed two peaks, which appeared at 0.95 and 1.17 V at bare GCE in 50 nM Ru1 and 50 mM TPA. The ECL peak at 0.95 V is mainly attributed to the radical TPA• + (formed during TPA oxidation), which can oxidize Ru(bpy)3+ (formed from the reduction of Ru(bpy)32+ by the TPA• free radical) to produce Ru(bpy)32+*, while the ECL peak at 1.17 V is generated from the conventional ECL route described above.34,35 At PGE, one ECL peak at 1.20 V was observed and its relative maximum ECL intensity (the ratio of ECL intensity on electrode area) is 2.7-fold greater than that at the GCE (see Figure S-5 in the Supporting Information). This could be explained by two possible reasons: (1) the hydrophobicity of the PGE surface is increased (Figure S-6 in the Supporting Information), which promotes the deprotonation of the TPA cation radicals so that the ECL efficiency at 0.95 V is reduced;36 and (2) the roughness of the PGE surface is increased, resulting in the increase of the real electrode area of the PGE surface (see Figure S-7 in the Supporting Information), and further resulted in the enhancement of the peak current.37,38 The results indicate that a high sensitivity for Ru1 is achieved at PGE. Therefore, PGE was chosen as a base electrode in this work. 3.2.2. AuNPs/Nafion/PGE. Figure 3 shows ECL intensity versus potential curves obtained at different electrodes after the ECL bioconjugated magnetic beads were incubated with 20 pg/ mL PSA. The ECL intensity at Nafion/GCE (Figure 3b) is 2.6fold greater than that obtained at the bare GCE (Figure 3a). This indicates that Nafion can concentrate the cleaved Ru1 part of the peptide. The ECL intensity at AuNPs/Nafion/GCE (Figure 3c) is 1.9-fold greater than that at Nafion/GCE (Figure 3b). This indicates that AuNPs can enhance the ECL intensity of the cleaved Ru1 part of the peptide on Nafion/GCE. This is to say, the ECL intensity at AuNPs/Nafion/GCE is 5.2-fold greater than that of the GCE. In addition, AuNPs were welldispersed into Nafion onto GCE, as shown via TEM analysis (see Figure S-8 in the Supporting Information). The ECL intensity at the AuNPs/Nafion/PGE (Figure 3f) was 1.8-fold greater than that obtained at a Nafion/PGE (Figure 3e) and

6.4-fold greater than that at PGE (Figure 3d), respectively. This indicates that a high sensitivity at the AuNPs/Nafion/PGE is achieved for the ECL detection of PSA compared to that at bare PGE. This enhanced sensitivity is attributed to the amplification of AuNPs39 and the ion exchange selectivity coefficients (106−107) of Nafion for cation Ru1.40 3.3. Analytical Performance for PSA. Important experimental parameters including the cleavage time, cleavage temperature, and preconcentration time for the determination of PSA using AuNPs/Nafion/PGE were optimized. The following conditions were chosen for the experiments: cleavage time, 40 min; cleavage temperature, 30 °C; and preconcentration time, 25 min (see Figure S-9 in the Supporting Information). Figure 4 shows ECL intensity versus potential profiles at AuNPs/Nafion/PGE under the optimal conditions. The ECL intensity increases as the concentration of PSA increases from 5.0 × 10−13 g/mL to 1.0 × 10−10 g/mL, while the increased ECL intensity is directly proportional to the concentration of PSA in the range from 5.0 × 10−13 g/mL to 3.0 × 10−11 g/mL. The linear regression equation can be expressed as ΔI = 326 + 107C (the units for C are pg/mL), and the correlation coefficient is 0.9813. The detection limit is 8 × 10−14 g/mL PSA (3σ),41 which is much lower than that of the previously reported method (see Table S-1 in the Supporting Information). The cutoff value for PSA in the diagnostic of prostate cancer is 4.0 ng/mL, while the value is much lower in the post-surgery recurrence42,43 and as a breast cancer screening target.44 For bare PGE, the ECL intensity increased with the increase of concentration of PSA from 8.0 × 10−12 g/ mL to 1.0 × 10−9 g/mL, while the increased ECL intensity was directly linearly proportional to the PSA concentration in the range from 1.0 × 10−11 g/mL to 1.0 × 10−10 g/mL (see Figure S-10 in the Supporting Information). The linear regression equation was ΔI = 206 + 6.3C (again, the units for C are pg/ mL) and the correlation coefficient was 0.9946. The detection limit was 5.0 × 10−12 g/mL PSA. Significantly, at AuNPs/ Nafion/PGE, a high sensitivity and a low detection limit were obtained. All results indicate that the good performance of the proposed bioanalytical system is achieved using two models on both bare PGE and AuNPs/Nafion/PGE. D

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indicate that the developed method may be competent for PSA assay in clinical applications.

4. CONCLUSIONS The present work describes the development of an ECL bioanalytic system based on the biocleavage of a ECL peptide probe and homogeneous detection for the first time for highly sensitive quantification of proteases. This system allows sensitive detection of PSA by conducting the cleavage and transduction steps at different surfaces, which diminish complicated electrode process and the reproducibility of the biosensor surfaces. A simple method for synthesis of ECL magnetic beads was developed through solid-phase synthesis, providing a feasible, simple, and wide-useful means for the synthesis of magnetic nanoprobe. The detection model with bare electrode is simple and fast while the detection model with AuNPs/Nafion/PGEs is more sensitive as an effective preconcentration of Nafion and an amplification of AuNPs. The proposed system may open a door to ultrasensitive ECL biosensing for other proteases and other detection techniques, including optics and electrochemistry.



ASSOCIATED CONTENT

S Supporting Information *

Experimental section, 12 figures, and two tables as noted in the text. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b01396.

Figure 4. (A) ECL intensity versus potential profiles of the ECL bioconjugated magnetic beads after reacted with different concentrations of PSA: blank (curve a), 0.5 pg/mL (curve b), 1.0 pg/mL (curve c), 3.0 pg/mL (curve d), 5.0 pg/mL (curve e), 8.0 pg/mL (curve f), 10 pg/mL (curve g), 15 pg/mL (curve h), 20 pg/mL (curve i), 30 pg/mL (curve j), 50 pg/mL (curve k), and 100 pg/mL (curve l). (B) Relationship of increased ECL intensity with concentration of PSA (inset shows the calibration curve of PSA). [In 0.10 M PBS (pH 7.4) containing 50 mM TPA at AuNPs/Nafion/PGE; scan rate = 50 mV/s and preconcentration time = 25 min.]



AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86-29-81530726. Fax: +86-29-81530727. E-mail: [email protected] (H. L. Qi). *Tel.: +86-29-81530726. Fax: +86-29-81530727. E-mail: [email protected] (C. X. Zhang).

In addition, it is worth-mentioned that AuNPs/Nafion/PGE exhibits outstanding reproducibility under continuously scanned for 10 cycles by monitoring ECL response in 0.10 M PBS (pH 7.4) containing 50 mM TPA with a relative standard derivation (RSD) of 4.1% for 15 pg/mL PSA (see Figure S-11 in the Supporting Information). This is attributed to the large ion exchange selectivity coefficients (106−107) for hydrophobic ions.40 A RSD of 5.4% was obtained using six AuNPs/Nafion/ PGEs for six measurements of 0.5 pg/mL PSA. Therefore, a good reproducibility using AuNPs/Nafion/PGEs is achieved. We also examined the selectivity of the ECL method toward PSA by testing the influence of other proteases (50 pg/mL) including thrombin, trypsin, MMP-2, and MMP-7. A remarkable change in the ECL signal was observed for 20 pg/mL PSA while negligible changes in the ECL intensity were observed in the presence of 2.5-fold higher concentrations of other tested proteases (see Figure S-12 in the Supporting Information). This result reflects the high selectivity of the peptide with the sequence CHSSKLQK associated with the effective enriching. The potential applicability of the proposed method in real samples was examined by assaying PSA in eight human serum samples, provided by Xi’an Friendship Medical Inspection (see Table S-2 in the Supporting Information). This ECL bioassay is demonstrated by good correlations with chemiluminescence immunoassay, using a MAGLUMI 4000 Automated Chemiluminescence Immunoassay (Snibe Co., Ltd, China). The data

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS Financial support from the National Science Foundation of China (Nos. 21475082, 21375084, 21275095) and the 111 Project (No. B14041) is gratefully acknowledged.



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DOI: 10.1021/acs.analchem.5b01396 Anal. Chem. XXXX, XXX, XXX−XXX

Electrogenerated Chemiluminescence Bioanalytic System Based on Biocleavage of Probes and Homogeneous Detection.

A novel electrogenerated chemiluminescence (ECL) bioanalytic system based on biocleavage of a ECL probe and homogeneous detection was designed and uti...
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