Ultrasensitive Protease Sensors Using Selective Affinity Binding

Nov 30, 2016 - The new sensor scheme using no washing process is compared with the new sensor ... (AP) group at the C-terminal of Gly-Pro-Arg (GPR-AP)...
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Ultrasensitive Protease Sensors Using Selective Affinity Binding, Selective Proteolytic Reaction, and Proximity-Dependent Electrochemical Reaction Seonhwa Park,‡ Gamwoo Kim,‡ Jeongwook Seo, and Haesik Yang* Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University, Busan 46241, Korea S Supporting Information *

ABSTRACT: The development of a fast and ultrasensitive protease detection method is a challenging task. This paper reports ultrasensitive protease sensors exploiting (i) selective affinity binding, (ii) selective proteolytic reaction, and (iii) proximity-dependent electrochemical reaction. The selective affinity binding to capture IgG increases the concentration of the target protease (trypsin as a model protease) near the electrode, and the selective proteolytic reaction by trypsin increases the concentration of the redox-active species near the electrode. The electrochemical reaction, which is more sensitive to the concentration of the redox-active species near the electrode than to its bulk concentration, provides an increased electrochemical signal, which is further amplified by the electrochemical−chemical redox cycling. An indium−tin oxide electrode modified with reduced graphene oxide, avidin, and biotinylated capture IgG is used as the electrode, and p-aminophenol liberated from an oligopeptide is used as the redox-active species. The new sensor scheme using no washing process is compared with the new sensor scheme using washing process, and with the conventional scheme using only proteolytic reaction. The new scheme provides a higher signal-to-background ratio and a lower detection limit. Moreover, the increased electrochemical signal offers a more selective protease detection. Trypsin can be detected in phosphate-buffered saline and in artificial serum containing L-ascorbic acid with a low detection limit of 0.5 pg/mL, over a wide range of concentrations, and with an incubation period of only 30 min without washing process. The washing-free electrochemical protease sensor is highly promising for simple, fast, ultrasensitive, and selective point-of-care testing of lowabundance proteases.

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chemical biosensors using both selective affinity binding and selective proteolytic reaction have been never attempted. In electrochemical detection, signals depend on the concentration of a redox-active species near an electrode surface rather than on its average concentration in solution (bulk concentration), because the electrochemical reaction occurs exclusively at its surface. In order to achieve a significant change in the electrochemical signal, the proteolytic generation or removal of a redox-active species should preferably occur near the electrode surface rather than in the bulk solution. For this purpose, the proteolytic removal of a redox-active species from the electrode surface has been reported.9,17−19 Although this method does not require washing procedures, the “signaloff scheme” has a limitation in lowering the sensitivity. Very importantly, the proteolytic generation of a redox-active species near the electrode surface can provide the “signal-on scheme,” and its combination with the affinity binding of a target protease to the electrode surface allows high sensitivity and washing-free detection procedure. However, such a combina-

he development of a simple protease detection method with a low detection limit, especially a washing-free, fast, and ultrasensitive assay, is a challenging task.1−3 Although numerous biosensors for protease detection have been developed, such a goal has not yet been achieved. These biosensors are classified into (i) immunosensors, based on the selective affinity binding between a target protease and a probe antibody,4−7 and (ii) protease sensors, based on the selective proteolytic reaction of a target protease.8−12 Immunosensors (in general, sandwich-type immunosensors) provide sensitive detection via high signal amplification within a short period of time; however, they require complicated washing or separation steps to remove the unbound labeled antibody.4−7 On the contrary, protease sensors can allow washing-free one-step procedures but are less sensitive than immunosensors because of their limited signal amplification.8−12 Interestingly, biosensors based on both selective affinity binding and selective proteolytic reaction have also been developed.13−16 The affinity binding was exploited to capture the target protease on a magnetic bead13−15 or microplate15,16 prior to its proteolytic reaction. However, such biosensors also require washing steps that complicate the detection procedure. Moreover, electro© XXXX American Chemical Society

Received: August 19, 2016 Accepted: November 15, 2016

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DOI: 10.1021/acs.analchem.6b03255 Anal. Chem. XXXX, XXX, XXX−XXX

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EXPERIMENTAL SECTION Chemicals and Solutions. A trypsin substrate (an oligopeptide containing a p-aminophenol (AP) group at the C-terminal of Gly-Pro-Arg (GPR-AP)) was obtained from Anygen Co., Ltd. (Jangseong-gun, Korea). Trypsin from porcine pancreas (type IX-S), pepsin from porcine gastric mucosa, papain from papaya latex, bovine serum albumin (BSA), human serum albumin (HSA), IgG from mouse serum, lysozyme from chicken egg white, AP, tris(2-carboxyethyl)phosphine (TCEP) hydrochloride, L-ascorbic acid (AA), ascorbate oxidase (AOx), uric acid (UA), acetaminophen (AcP), and all the reagents used for the preparation of buffer solutions and electrode pretreatments were obtained from Sigma-Aldrich Co. A graphene oxide (GO)-dispersed aqueous solution was obtained from Graphene Laboratories Inc. (Calverton, NY, USA). Biotinylated antitrypsin IgG (60R1644) was purchased from Fitzgerald, Inc. (Acton, MA, USA). A phosphate-buffered saline (PBS, pH 7.4) solution and 1% (w/v) BSA were used to prepare a PBSB solution. Artificial serum comprised PBS, 1% (w/v) HSA, 0.1 mM AA, 0.1 mM UA, and 0.1 mM AcP. ITO electrodes were obtained from Corning Co. (Daegu, Korea). Preparation of Sensing Electrodes. ITO electrodes were pretreated with a 5:1:1 solution of H2O, H2O2 (30%), and NH4OH (30%) at 70 °C for 1 h.26 To prepare partially GOmodified ITO (GO/ITO) electrodes, each pretreated ITO electrode was immersed in 400 μL of an aqueous solution containing 50 μg/mL GO for 2 h at 25 °C, followed by washing with distilled water. rGO/ITO electrodes were obtained by applying −1.0 V to the GO/ITO electrodes for 100 s in 0.5 M NaCl (Figure S-1 of the Supporting Information).22 To obtain avidin-modified rGO/ITO (avidin/rGO/ITO) electrodes, 70 μL of a carbonate buffer solution (20 mM, pH 9.6) containing 10 μg/mL avidin was dropped onto the rGO/ITO electrodes for 2 h at 20 °C. Next, 70 μL of a PBSB solution was dropped to obtain BSA/avidin/rGO/ITO electrodes. Biotinylated antitrypsin IgG was immobilized by dropping 70 μL of a PBSB solution containing 10 μg/mL biotinylated antitrypsin IgG onto the BSA/avidin/rGO/ITO electrodes. The final sensing electrodes were maintained in the treated state for 30 min at 4 °C. Procedure for Trypsin Detection. Solutions of TCEP (8.0 mM) and GPR-AP (0.4 mM) were prepared in PBS. Solutions containing different concentrations of trypsin were prepared in PBS and artificial serum. In Scheme 1a, 70 μL of PBS solutions containing different concentrations of trypsin was dropped onto a sensing electrode for 30 min at 4 °C, and the sensing electrodes were then washed with PBSB. Subsequently, 250 μL of the GPR-AP solution (0.4 mM in PBS), 250 μL of the TCEP solution (0.8 mM in PBS), and 500 μL of a PBS solution were mixed and injected into an electrochemical cell containing the sensing electrode on which trypsin was immobilized. The electrochemical cells were incubated at 37 °C for 30 min. Total detection period was 60 min. In Scheme 1b, 250 μL of PBS solutions containing different concentrations of trypsin was mixed with 250 μL of the GPR-AP solution (0.4 mM in PBS), 250 μL of the TCEP solution (0.8 mM in PBS), and 250 μL of a PBS solution, and the mixture was injected into an electrochemical cell containing a BSA/avidin/rGO/ITO electrode. The electrochemical cells were incubated at 37 °C for 30 min. Total detection period was 30 min. In Scheme 1c, 250 μL of PBS solutions (or artificial

tion has not yet been investigated for the electrochemical detection of proteases. In recent years, we have developed protease sensors based on proteolytic reactions and electrochemical signal amplification.20−22 In all cases, however, selective affinity binding was not employed, and the proteolytic reaction occurred in the bulk solution. The redox cycling, i.e., the repeated cycle of heterogeneous electrochemical reaction and homogeneous chemical reaction(s), was used to obtain high electrochemical signals, and it was employed in immunosensors,23 microRNA biosensors,24 and cell-based biosensors.25 Avidin-modified indium−tin oxide (ITO) electrodes were used to obtain both low nonspecific adsorption of proteases and low background levels.22 Here, we report a fast and ultrasensitive electrochemical protease sensors that take advantage of selective affinity binding, selective proteolytic reaction, and proximity-dependent electrochemical reaction. This combination allows a high electrochemical signal, which is further amplified using electrochemical-chemical (EC) redox cycling at an ITO electrode modified with reduced graphene oxide (rGO) and avidin.18,19 Avidin is used to immobilize a biotinylated capture IgG and to obtain low nonspecific binding. To demonstrate the superiority of the new sensors, three detection schemes for trypsin as a model protease are compared: (1) new trypsin detection using affinity binding, washing process, and proteolytic reaction (Scheme 1a), (2) conventional washingfree trypsin detection using only proteolytic reaction20−22 (Scheme 1b), and (3) new washing-free trypsin detection using both affinity binding and proteolytic reaction (Scheme 1c). Scheme 1. Schematic of Three Electrochemical Trypsin Detection Methods: (a) New Trypsin Detection Using Affinity Binding, Washing Process, and Proteolytic Reaction, (b) Conventional Washing-Free Trypsin Detection Using Proteolytic Reaction, and (c) New Washing-Free Trypsin Detection Using Both Affinity Binding and Proteolytic Reaction

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Analytical Chemistry serum) containing different concentrations of trypsin was mixed with 250 μL of the GPR-AP solution (0.4 mM in PBS), 250 μL of the TCEP solution (0.8 mM in PBS), and 250 μL of a PBS solution. In the case of trypsin detection in artificial serum, 100 μL of a PBS solution containing 10 U/mL AOx and 150 μL of a PBS solution were used instead of 250 μL of a PBS solution. The mixture was injected into an electrochemical cell containing the sensing electrode. The electrochemical cells were incubated at 37 °C for 30 min. Total detection period was 30 min. The final concentrations of TCEP and GPR-AP were 2.0 and 0.1 mM, respectively. Electrochemical Measurements. Teflon electrochemical cells were assembled using the sensing electrode, a Ag/AgCl (3 M NaCl) reference electrode, and a platinum counter electrode. The exposed geometric area of the sensing electrode was approximately 0.28 cm 2. Chronocoulometry and cyclic voltammetry were performed at 37 °C using a CHI 650E or CHI 405A (CH Instruments, Austin, TX, USA).

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RESULTS AND DISCUSSION To obtain a high signal-to-background ratio in EC redox cycling as shown in Scheme 1, the selection of an appropriate

Figure 2. (a) Comparison of the charge values measured at 100 s in the chronocoulograms (Figure S-2) obtained using Scheme 1 at three trypsin concentrations (0, 1 ng/mL, and 1 μg/mL). (b) Comparison of the charge values measured at 100 s in the chronocoulograms obtained using Scheme 1c in a blank PBS solution and PBS solutions containing 100 ng/mL trypsin, pepsin, papain, lysozyme, IgG, or BSA.

potential range where the redox-active species liberated by proteolytic reaction (trypsin product) undergoes fast EC redox cycling. The reductant should rapidly react with the oxidized form of the redox-active species and should not decrease trypsin activity. Our previous reports show that the use of rGO/ITO as an electrode, AP as a redox-active species, and TCEP as a reductant meets these requirements.20,22 At the outset, cyclic voltammograms were recorded to confirm the high signal-to-background ratio in EC redox cycling using the avidin/rGO/ITO electrode, AP, and TCEP (Figure 1a). The quasi-reversible redox peaks of AP having a formal potential of ca. 0.06 V were observed at the avidin/rGO/ITO electrode because of the high electrocatalytic activity of rGO (curve i of Figure 1a).22 In a solution containing GPR-AP and TCEP, anodic faradaic currents were negligible up to 0.3 V (curve ii of Figure 1a), indicating that the electrooxidation of GPR-AP and TCEP is slow up to this potential. This result also shows that the contribution of the faradaic currents of GPR-AP and TCEP to the background level is negligible in the potential region up to 0.3 V. In the presence of trypsin in solution, substantial faradaic currents were observed from 0.0 V because AP liberated by trypsin from GPR-AP participated in EC redox cycling (curve iii of Figure 1a). The anodic faradaic currents were much higher than those generated by only AP (curve i of Figure 1a) because of the fast EC redox cycling. The voltammetric data confirm that this EC redox cycling generates a high signal-to-background ratio. In protease detection, chronocoulograms were used instead of cyclic voltammograms. In order to determine the applied potential that provides the highest signal-to-background ratio, the charges in the presence

Figure 1. (a) Cyclic voltammograms obtained (at a scan rate of 50 mV/s) at avidin/rGO/ITO electrodes after an incubation period of 30 min at 37 °C in PBS containing (i) 0.1 mM AP, (ii) 0.1 mM GPR-AP and 2.0 mM TCEP, and (ii) 0.1 mM GPR-AP, 2.0 mM TCEP, and 10 μg/mL trypsin. (b) Histogram of the signal-to-background (S/B) ratios calculated from the charge values measured at 100 s in the chronocoulograms obtained at avidin/rGO/ITO electrodes at four applied potentials (0.05, 0.10, 0.15, and 0.20 V) after an incubation period of 30 min at 37 °C in PBS containing (i) 0.1 mM GPR-AP and 2.0 mM TCEP and (ii) 0.1 mM GPR-AP, 2.0 mM TCEP, and 10 μg/ mL trypsin.

electrode, redox-active species, and reductant is of primary importance. The electrode should offer fast electro-oxidation of a redox-active species along with slow electro-oxidation of a reductant. The redox-active species connected to an oligopeptide (trypsin substrate) should be redox-inactive in the C

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Figure 4. (a) Chronocoulograms obtained using Scheme 1c after an incubation period of 30 min at 37 °C in a mixed solution of PBS containing 0.1 mM GPR-AP, 2.0 mM TCEP, 10 U/mL AOx, and artificial serum containing different concentrations of trypsin. (b) Calibration plot of the charge measured at 100 s in the chronocoulograms of panel a.

washing process. Finally, the proteolytic reaction and electrochemical measurements were performed in a solution containing GPR-AP and TCEP. The captured trypsin cleaved the peptide bond of GPR-AP, liberating AP. After a 30 min incubation for proteolytic reaction, the application of a potential of 0.15 V oxidized AP to p-quinone imine (QI), which was reduced back to AP by TCEP. The regenerated AP was reoxidized to QI. This EC redox cycling allows high signal amplification. In Scheme 1b and c, the proteolytic reaction and electrochemical measurements were performed in a mixture containing trypsin, GPR-AP, and TCEP, without a washing process. In Scheme 1c, affinity binding of trypsin as well as proteolytic reaction occurred during the 30 min incubation. In Scheme 1b, the concentration of AP near the electrode is similar to the bulk concentration. However, in Scheme 1c, the AP concentration near the electrode is much higher than the bulk concentration, because of the captured trypsin near the electrode. Thus, the detection process in Scheme 1c provides higher electrochemical signals than that in Scheme 1b. Figure S-2 shows chronocoulograms obtained for different concentrations of trypsin using the three detection schemes. In all the schemes, the measured charge increased with increasing trypsin concentration. In the absence of trypsin, similar charge values were obtained at 100 s (Figure 2a), indicating that the background levels are similar in the three schemes. At trypsin concentrations of 1 ng/mL and 1 μg/mL (Figure 2a), the charge for Scheme 1c was much higher than that for Scheme 1b and slightly higher than that for Scheme 1a, indicating that the signal level for Scheme 1c is the highest. Importantly, only

Figure 3. Calibration plots of the charge measured at 100 s in the chronocoulograms (Figure S-2) obtained using (a) Scheme 1 part a, (b) b, and (c) c after an incubation period of 30 min at 37 °C in PBS containing 0.1 mM GPR-AP, 2.0 mM TCEP, and different concentrations of trypsin. All experiments were conducted using 3 different electrodes for each sample. The data were subtracted from the mean value at a concentration of zero determined from seven measurements. The dashed line corresponds to three times the standard deviation (SD) of the charge at zero concentration. The error bars represent the SD of three measurements.

of GPR-AP and TCEP and in the presence of GPR-AP, TCEP, and trypsin were obtained at four applied potentials (Figure 1b). The highest signal-to-background ratio was observed at 0.15 V, which was used as the applied potential for further experiments. The purpose of this study is to investigate the usefulness of electrochemical trypsin detection using both affinity binding and proteolytic reaction (Scheme 1a and c). For comparison, conventional trypsin detection using only proteolytic reaction (Scheme 1b) was also performed. In Scheme 1a, trypsin in a sample solution was captured on an antitrypsin IgGimmobilized sensing electrode. Uncaptured trypsin and interfering redox-active species were then removed via a D

DOI: 10.1021/acs.analchem.6b03255 Anal. Chem. XXXX, XXX, XXX−XXX

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detected over a wide range of concentrations from 0.5 pg/mL to 10 μg/mL. This result indicates that Scheme 1c is practically appealing.

affinity-bound trypsin near the electrode contributes to the proteolytic reaction in Scheme 1a, whereas only unbound trypsin in the solution contributes in Scheme 1b. On the other hand, in Scheme 1c, both affinity-bound trypsin near the electrode and unbound trypsin in the solution contribute to the proteolytic reaction, thus explaining the highest signal level observed in this case. The comparison clearly shows that Scheme 1 parts a and c are better than Scheme 1b for achieving a high signal-to-background ratio. Other proteases may slowly cleave GPR-AP even in the absence of trypsin. Because Scheme 1c uses both affinity binding and proteolytic reaction, the contribution of the undesired cleavage to the total charge is small compared to that in Scheme 1b. In Figure 2b, the charge obtained in a blank solution was compared with the charges obtained in solutions containing 100 ng/mL trypsin, pepsin, papain, lysozyme, IgG, or BSA. Except for trypsin, the charge values were found to be similar to that of the blank solution. Moreover, the charge measured for the trypsin solution was much higher than that of the blank solution. This result shows that the selective affinity binding as well as the selective proteolytic reaction increases the sensor selectivity. Figure 3 shows calibration plots obtained from the chronocoulograms in Figure S-2. As summarized in Table S1, Scheme 1c offers better simplicity and shorter detection period as well as lower detection limit. The calculated detection limits for Scheme 1 parts a, b, and c were approximately 1 pg/ mL, 0.8 ng/mL, and 0.5 pg/mL, respectively. Trypsin detection using both affinity binding and proteolytic reaction (Scheme 1a and c) shows much lower detection limits and wide detection ranges up to 10 μg/mL. The detection limit of 0.5 pg/mL is the lowest among the reported values for trypsin (Table S-2). Moreover, the detection limit of 1 pg/mL for Scheme 1a was much lower than that previously obtained using the scheme similar to Scheme 1a (optical detection using both affinity binding and proteolytic reaction),13−16 because the EC redox cycling at rGO/ITO electrode allows a high signal-tobackground ratio and because the electrochemical reaction is more sensitive to the concentration of AP near the electrode than to its bulk concentration. Very importantly, the total detection periods for Scheme 1 parts a, b, and c (excluding the periods required for preparing sensing electrodes) were 60, 30, and 30 min, respectively. Scheme 1c allows a lower detection period because the detection was carried out without washing process. Nevertheless, Scheme 1c is the most sensitive. Clearly, Scheme 1c offers a simple, fast, and ultrasensitive trypsin detection method effective over a wide range of concentrations. To investigate the possibility of the practical application of Scheme 1c, trypsin detection in artificial serum was performed. Because real serum contains trypsin inhibitors, artificial serum containing AA, UA, and AcP was employed instead. Although AA is the most problematic interfering redox-active species, AOx could rapidly oxidize AA to a redox-inactive species. Therefore, addition of AOx to the artificial serum minimized the interference of AA (Figure S-3a). Moreover, the background level was not affected by other interfering redox-active species such as UA and AcP (Figure S-3a). The signal-tobackground ratio observed at 0.15 V in the artificial serum (57.8, Figure S-3b) was similar to that in the PBS solution (63.1, Figure 1b). Figure 4a shows chronocoulograms obtained for various concentrations of trypsin spiked in artificial serum, and Figure 4b displays the corresponding calibration plot. The calculated detection limit was also 0.5 pg/mL, and trypsin was

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CONCLUSIONS We developed ultrasensitive protease sensors that combines the advantages of selective affinity binding, selective proteolytic reaction, and proximity-dependent electrochemical reaction. The selective affinity binding of trypsin and the selective proteolytic reaction by the bound trypsin increased the concentration of liberated AP near the electrode, which in turn provided a highly enhanced electrochemical signal. The EC redox cycling involving rGO/ITO electrode, AP, and TCEP further amplified the electrochemical signal. In terms of signal amplification and sensitivity, the new sensor scheme using affinity binding and proteolytic reaction (Scheme 1 parts a and c) was better than the conventional scheme using only proteolytic reaction (Scheme 1b). Moreover, the enhanced electrochemical signal provides a more selective protease detection. Trypsin in PBS and in artificial serum can be detected using Scheme 1c with a detection limit of 0.5 pg/mL and with an incubation period of only 30 min. The developed detection schemes can be readily applied to the detection of low-abundance proteases.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b03255. More supporting data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions ‡

S.P. and G.K. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Research Foundation of Korea (2015R1A2A2A01002695, 2016M3A7B4910538, and 2014H1A8A1020279). This material is also based upon work supported by the Ministry of Trade, Industry & Energy (MOTIE, Korea) under Industrial Technology Innovation Program No. 10062995.

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