Portable, Self-Powered and Light-Addressable Photoelectrochemical

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Portable, Self-Powered and Light-Addressable Photoelectrochemical Sensing Platforms using pH Meter Readouts for HighThroughput Screening of Thrombin Inhibitor Drugs Juan Wang, Mengmeng Song, Chengguo Hu, and Kangbing Wu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01979 • Publication Date (Web): 12 Jul 2018 Downloaded from http://pubs.acs.org on July 12, 2018

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Analytical Chemistry

Portable, Self-Powered and Light-Addressable Photoelectrochemical Sensing Platforms using pH Meter Readouts for High-Throughput Screening of Thrombin Inhibitor Drugs Juan Wang,a Mengmeng Song,b Chengguo Hu,*b Kangbing Wu*a a

Key Laboratory for Material Chemistry of Energy Conversion and Storage, Ministry of Education,

School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China b

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China

* To whom correspondence should be addressed:

Dr. Chengguo Hu, Email: [email protected]; Prof. Kangbing Wu, Email: [email protected] (K. Wu).

ABSTRACT: In this work, a self-powered, portable and light-addressable photoelectrochemical sensor (P-LAPECS) is developed for efficient drug screening using a hand-held pH meter readout. The sensor which employs thrombin inhibitors as the drug model is constructed by evenly immobilizing biotin-labeled and thrombin-cleavable peptides on eight separated sensing zones of a single gold film electrode. The incubation of each peptide sensing zone with thrombin leads to the reduction of binding sites for streptavidin-labeled fullerene (C60) PEC bioprobes, which directly reflects the activity of thrombin by the variation of both photocurrent and photovoltage, and therefore allows the screening of thrombin inhibitors using either a single-channel electrochemical analyzer or a portable pH meter. In consequence, the inhibition efficiency evaluation of multiple thrombin inhibitors can be achieved by just one electrode, and the screening result obtained by the pH meter is very close to that acquired by the electrochemical analyzer. Moreover, P-LAPECS can realize the light-addressable detection of thrombin ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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with detection limit as low as 0.05 pM. The present work thus demonstrates the possibility of constructing portable, inexpensive, sensitive and high-throughput biosensing platforms using ubiquitous pH meters for labs all over the world. KEYWORDS: photoelectrochemical sensors; self-powered; light-addressable; pH meter; thrombin inhibitors.

INTRODUCTION Photoelectrochemical sensors are a kind of rapidly developed analytical technique, which not only possess the merits of high integration and low cost resembling traditional electrochemical sensors, but also exhibit high sensitivity due to the difference of excitation and detection signals.1-3 At present, PEC sensors have been used for the detection of various enzymes and their inhibitors, such as protein kinase,4 acetylcholinesterase5 and thrombin.6-7 Unfortunately, all these works are performed using single-channel PEC sensing systems with limited detection throughput, which are not suitable for high-throughput screening of enzyme-targeted drugs. To overcome this limitation, several sensing principles, such as electrode arrays,8-9 multiple tags,10-11 tuned potentials,12-13 and double-channel PEC mode,14 have been proposed for multiplexed PEC biosensing. Recently, we also proposed a light-addressable detection strategy to realize the multiplexed PEC detection of DNA sequences or tumor markers using just one working electrode.15-16 However, the employment of traditional three-electrode cells and electrochemical analyzers limits more or less the development of portable PEC sensing systems for cost-effective on-site assays. Portable electronic devices have revolutionized current diagnostic modes by enabling point-of-care testing of many diseases, which can effectively solve the problems of high cost and geographic restrictions for the existing biomedical testing technology.17 Taking into account the advantages of ACS Paragon Plus Environment

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portable size, low cost and easy operation, electrochemical or electric detectors such as glucose meters, pH meters and digital multimeters are widely employed for biomedical assays. For instance, many groups have taken advantage of glucose meters to detect a series of non-glucose targets.18-19 Similarly, pH meters have also been successfully applied to the detection of cardiac markers17 and DNA20 based on the change of solution pH during biological reactions. In the field of PEC sensing, the most commonly used portable devices are digital multimeters,8,21-22 which however generally need to be used in combination with an external capacitor. In addition, it is well known that pH meter can measure the voltage difference between two electrodes and therefore may be a potential signal readout of self-powdered PEC sensing systems. In this work, we demonstrate that pH meters are also a very promising signal detector of PEC sensors for high-throughput biosensing. We constructed a portable and light-addressable photoelectrochemical sensor (P-LAPECS) for drug screening on a single electrode (Figure 1), which in combination with a platinum (Pt) wire counter electrode forms a self-powered PEC sensing system resembling solar cells. And the thrombin inhibitor, which is vastly demanded for discovery of thrombolytic drugs and clinical treatment of other coagulant diseases,23-24 was selected as the screening model. Specifically, the sensor was fabricated via a four-step process: (i) evenly immobilization of biotin-labeled and thrombin-cleavable peptides on different areas of an eight-zone gold film electrode, (ii) incision of the peptides with thrombin (inhibited by different inhibitors), (iii) reaction of a streptavidin-labeled C60-based PEC bioprobe, and (iv) light addressable screening of multiple inhibitors one by one via a traditional electrochemical analyzer or a portable pH meter. The addressing photovoltaic signals produced by a handheld red laser pen (650 nm, 150 mW) were proportional to the amount of the bioprobe, corresponding to the uncleaved biotin-labeld peptide, which in turn depended on the activity

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of thrombin. Benefiting from the conveniently improved detection throughput of LAPECS,25-26 a single eight-zone P-LAPECS can not only achieve screening of thrombin inhibitors but also obtain a full calibration plot of thrombin (concentration ranging from 0.1 pM to 1.0 nM). Therefore, the combination of integrated light-addressable PEC sensing systems with portable signal detection devices enables the construction of portable, inexpensive, sensitive and high-throughput PEC sensing systems for on-site multiplexed bioassay or drug screening.

Figure 1. Schematic representation for the screening of thrombin inhibitors (lower four sensing areas) and detection of thrombin (upper four sensing areas) using a single eight-zone P-LAPECS.

EXPERIMENTAL SECTION Chemicals Ascorbic acid (AA), chloroauric acid (HAuCl4·4H2O), hydroxylammonium chloride (NH2OH·HCl), trisodium citrate dehydrate (cit), sodium borohydride (NaBH4), disodium hydrogen phosphate (Na2HPO4·12H2O),

sodium

dihydrogen

phosphate

(NaH2PO4·2H2O),

potassium

ferricyanide

(K3Fe(CN)6), potassium ferrocyanide (K4Fe(CN)6), potassium chloride (KCl), hydrochloric acid (HCl), sodium hydroxide (NaOH), tris (hydroxymethyl) aminomethane (Tris), N-hydroxysuccinimide (NHS), 6-mercapto-1-hexanol (MCH), monoethanolamine (MEA) and Congo red (CR) were purchased from Sinopharm Chemical Reagent Co. Ltd., China. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) ACS Paragon Plus Environment

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and fullerene (C60) were the products of J&K Scientific Ltd., China. Thrombin and albumin from bovine serum (BSA) were obtained from Sigma-Aldrich. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl) was purchased from Shanghai Medpep Co., Ltd. Two substrate peptides for thrombin (i.e., biotin-GLVPR↓GSGGGLKC (peptide 1) and biotin-GGLVPR↓GSC (peptide 2)), streptavidin (SA), lysozyme and trypsin were obtained from Sangon Biotechnology Co. (Shanghai, China) and used without further purification. L-cysteine (L-cys), 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), benzamidine hydrochloride hydrate (Benzamidine), argatroban monohydrate (Argatroban) and phenylmethanesulfonyl fluoride (PMSF) were purchased from Aladdin Reagent Company (Shanghai, China). Polyimide films (PI) were the product of Dongguan Xinshi Packaging material Co. Ltd., China. Silver paste (BQ6880E) was the product of Uninwel, Co., Ltd. Epoxy glue was purchased from Hu'nan Magic Power Industrial Co., China. All aqueous solutions were prepared using ultrapure deionized water (> 18 MΩ·cm) produced on Heal Force, Nison Instrument Ltd., Shanghai, China. The electrolyte solution for all the photocurrent measurements was 0.1 M PBS (pH 7.4) containing 0.1 M AA. All the buffer solutions were adjusted to suitable pH values with 1.0 M HCl or NaOH by a pH meter (PB-10, Sartorius). Apparatus Scanning electron microscopy (SEM) images were collected on a field emission scanning electron microscope (Zeiss, Germany). High-resolution transmission electron microscopy (HRTEM) measurements were carried out using a FEI Tecnai G2 F30 transmission electron microscope. UV-Vis spectra were collected on a UV 2550 spectrophotometer (Shimadzu, Japan). A high speed vibrating ball-milling machine (QM-3B, Nanjing NanDa Instrument Plant, Nanjing, China) was employed to mill samples. Sheet resistance of films or coatings was measured with a four-point probe sheet resistance



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tester (ST-21H, China). All the photocurrent responses were collected by a CHI 830 analyzer at open circuit potentials in a three-electrode system, including a P-LAPECS working electrode, a platinum wire counter electrode and a saturated calomel reference electrode (SCE). A portable pH meter (PHB-1, Hangzhou Qiwei Instrument Co. Ltd., China) was used to record the photovoltage responses produced by the two-electrode system comprising a P-LAPECS working electrode and a platinum wire counter electrode. A red laser pen (150 mW, 650 nm) with an irradiation diameter of ~ 2.5 mm was used as the light source.

Preparation of C60/CR-L-cys@SA PEC Bioprobe. First of all, a ball-milling method was used to prepare the C60/CR nanohybrid. Briefly, the mixture of C60 and CR (mass ratio =1:3) was transferred to a 50-mL agate jar with agate balls at a ball-to-powder mass ratio of 20:1 and milled for 1 h. The resulting ball-milled mixture was dissolved in water and thoroughly washed with water on a mixed cellulose ester membrane (MCE, pore size 0.1 µm, Shanghai Xinya Purifier Devices Factory, China) by filtration until the filtrate became colorless. Then, the C60/CR aqueous solution was obtained by transferring the material along with the membrane to water, sonicating for several minutes and discarding the membrane. To obtain a better water-soluble C60/CR solution, the resulting C60/CR solution was centrifuged at 12000 rpm for 5 min and the supernatant was collected for further use. Then, a L-cys derived C60/CR composite (C60/CR-L-cys) for labeling of biomolecules was prepared according to a literature with slight modification.25 In brief, 0.20 g L-cys was dissolved in 0.60 mL NaOH solution (7.1 mol/L) and then the above C60/CR supernatant was added into the solution under stirring. After 2 days of stirring, the product C60/CR-L-cys was obtained through filtration and washed with water until the filtrate became neutral. For comparison, the synthesis of L-cys


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derived C60 by other approaches can be found in Supporting Information. Finally, a SA-labeled C60/CR-L-cys bioprobe (C60/CR-L-cys@SA) was prepared through the interaction between the active carboxylic group of L-cys and the amine groups on SA (Figure S1). Briefly, 1.0 mL of the resulting C60/CR-L-cys aqueous solution (calculated 1.0 mg/mL for C60) was diluted with 1.5 mL PBS (10.0 mM, pH 7.4) by sonication for 5 min, and mixed with 1.0 mL of a freshly prepared aqueous solution of 20.0 mg/mL EDC and 10.0 mg/mL NHS in a 5-mL plastic vial. The mixture reacted for 1.0 h under gentle shaking at room temperature on a multifunctional rotary table (QB-208, Qilinbeier, Haimen, China). Then, 500.0 µL of 0.1 mg/mL SA in 10.0 mM PBS (pH 7.4) was added and the reaction kept going at room temperature for additional 6.0 h. The resulting mixture solution was washed with 0.1 M PBS (pH 7.4) by centrifugation at 12000 rpm for 5 min, which was repeated for at least three times to remove any unreacted EDC, NHS and SA. The use of PBS with high ionic strength (0.1 M) ensures the efficient sedimentation of C60/CR-L-cys during centrifugation and avoids its possible mass loss during purification. The final product, i.e., C60/CR-L-cys@SA, was re-dispersed in 1.0 mL PBS (1.0 mM, pH 7.4) and stored at 4 oC under darkness for further use.

Construction of P-LAPECS A gold nanoparticle conductive layer coated PI film electrode (GPIFE) was prepared by an electroless plating method26 and used as the substrate electrode for fabricating the P-LAPECS, which was for the detection of thrombin and screening of its inhibitors using the light addressing strategy and the portable pH meter readout (Figure S2 and Figure S3). The corresponding experimental details are also presented in Supporting Information.



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RESULTS AND DISCUSSION Characterizations of C60/CR-L-cys. In our previous works,26 we demonstrated that a fullerene-based all-carbon photovoltaic material prepared by physically grinding C60 with Congo red and carboxylated multi-walled carbon nanotubes (i.e., MWNTCOOH-CR-C60) exhibited unique features for PEC biosensing, e.g., high sensitivity, good dispersion ability and favorable stability. However, the quality variation of carbon nanotubes from different batches or manufactures and the strong non-specific adsorption of proteins on MWNTCOOH27 may influence the reproducibility and selectivity of the resulting PEC biosensor. Here, we simplify the synthesis of C60-based PEC sensing materials by directly introducing derivable groups on C60 materials through a well-developed amino addition reaction method (Figure 2A).25,28 As indicated in Figure 2B, the direct addition of L-cys onto C60 by either chemical reactions or physical ball milling generally produces C60 materials with weak photocurrent responses (curves a and c in Figure 2B). In contrast, the ball-milling treatment of C60 with CR apparently improves the photocurrents (curves b and d in Figure 2B). Therefore, we firstly prepared the C60/CR nanohybrid by ball milling, and then reacted it with L-cys to introduce derivable carboxyl group for biolabeling. As expected, the resulting C60/CR-L-cys nanohybrid also possesses a large photocurrent of more than 60 µA (curves f in Figure 2B). Moreover, the UV-Vis absorption tendency of the above C60 nanomaterials (Figure 2C) are in good agreement with their photocurrent responses, which directly reflects the relationship between dispersion ability and PEC behaviors of C60 materials.


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Figure 2. Synthesis procedures (A), photocurrent responses (B) and UV-Vis spectra (C) of different C60-based PEC sensing materials: a. C60-L-cys; b. C60-L-cys/CR; c. C60/CR/L-cys; d. C60/CR; e. C60/CR (supernatant); f. C60/CR-L-cys. The concentration of C60 in all the nanohybrid solutions (inset of Part C) is calculated to be 1.0 mg/mL without considering the mass loss of C60 during synthesis. Photocurrent measurements were performed by dropping 2 µL of the C60 solutions on a GPIFE, drying under an infra-red lamp and illuminating with a red laser pen (150 mW, 650 nm). All the above C60 nanohybrid solutions for UV-Vis measurements were diluted for 50 times with water.



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SEM and TEM images indicate that the C60/CR nanohybrid prepared by the ball-milling method exhibits irregular granular structures (Figure S4A), and the further modification with L-cys hardly changes its morphology (Figure S4B). Moreover, after the overnight absorption onto the surface of GPIFE from their aqueous solutions, C60/CR-L-cys shows an obviously larger photocurrent than C60/CR (Figure S4C), which should arise from the formation of Au-S bond between C60/CR-L-cys and the gold film electrode and therefore proves the successful modification of C60/CR by L-cys. In this context, we tentatively examined the PEC biosensing performance of C60/CR-L-cys by covalently binding the goat anti-rabbit IgG onto C60/CR-L-cys for further immunoreaction with rabbit IgG that was immobilized on a GPIFE. The results indicate that the target rabbit IgG (1 ng/mL) adsorbed on the substrate electrode can produce a large photocurrent of about 15.5 µA after the immunoreaction, while the photocurrent response produced by a control interferent (i.e., 1 µg/mL CEA) is only 0.7 µA (Figure S5). Therefore, an efficient approach was established for the scalable synthesis of excellent C60-based PEC sensing materials through the ball milling treatments and amino additional reactions. It should be pointed out that the C60/CR nanohybrid purified by centrifugation in our previous reference26 possessed a rather weak photocurrent as compared with the material purified by filtration here (Figure S6A), which was ascribed to the loss of a large portion of soluble C60/CR during repeated centrifugation treatment (Figure S6B). In fact, the C60/CR nanohybrid prepared by just 1 h ball-milling treatment has a so good aqueous dispersion ability that a large portion of C60/CR remains stable even at a high centrifugation of 12000 rpm. Therefore, filtration washing on filter membranes with pore sizes no more than 0.1 µm is a preferred purification method for preparing C60/CR with good dispersion ability, high concentration and large photocurrents.


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Construction of P-LAPECS. To further examine the analytical performance of C60/CR-L-cys for PEC biosensing, we synthesized the C60/CR-L-cys@SA bioprobe for the detection of thrombin and screening of its inhibitors (Figure S2). In principle, C60/CR-L-cys@SA that was synthesized by covalently fixing SA onto C60/CR-L-cys with the aid of EDC and NHS can selectively bond to biotin-labeled and thrombin-cleavable peptide arrays on a GPIFE (Figure 3A). However, when the peptide arrays are incubated with thrombin or the mixture of thrombin and its inhibitors in advance, a portion of the peptides will be cleaved by thrombin, leading to

the

reduction

of

the

binding

site

for

C60/CR-L-cys@SA

and

the

decrease

of

photocurrents/photovoltages. As a result, the concentration/activity of thrombin or its inhibitors can be reflected by the variation of photocurrents or photovoltages.

Figure 3. (A) Schematic construction procedures and work principle of the sensor. (B) UV-Vis spectra of 0.1 mg/mL SA in 0.01 M PBS (pH 7.4) (red line) and the supernatant of the first centrifugation-based washing solution during the synthesis of C60/CR-L-cys@SA (black line). (C) Cyclic voltammograms in 1.0 M KCl containing 5.0 mM Fe(CN)63-/4of different electrodes during the fabrication of P-LAPECS: a. GPIFE; b. peptide/GPIFE; c. MCH/peptide/GPIFE; d. MEA/MCH/peptide/GPIFE; e. probe/MEA/MCH/peptide/GPIFE; f. thrombin (1 nM)/MEA/MCH/peptide/GPIFE; g. probe/thrombin/MEA/MCH/peptide/GPIFE. (D) SEM images of the above mentioned electrodes (a, b, e, g). Scale bars are 200 nm.



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As shown in Figure 3B, the strong UV-Vis absorption of SA at 260 nm almost disappears for the supernatant of the centrifuged C60/CR-L-cys@SA solution after amide reaction, which demonstrates the efficient modification of SA on C60/CR-L-cys. At the same time, the successful construction of the P-LAPECS is demonstrated by cyclic voltammetry (CV) (Figure 3C). Compared with those on the bared GPIFE (curve a), lower redox peak currents and larger peak-to-peak potential differences (∆Ep) were observed on voltammograms of the peptide modified GPIFE (peptide/GPIFE) (curve b), due to the repressed electron transfer rate of [Fe(CN)6]3/4- by insulative peptides. In addition, the successive incubation of the electrode with MCH (curve c), MEA (curve d) and C60/CR-L-cys@SA (curve e) causes the gradual decrease of the redox peak currents. On the contrary, the redox peak currents of the electrode (MEA/MCH/peptide/GPIFE) are partially recovered when the sensing arrays are incubated with thrombin (curve f), which reduces the surface coverage of the peptides and facilitates electron transfer. As expected, the further incubation of the thrombin-treated P-LAPECS with the bioprobe also causes a decrease of the redox peak currents (curve g). The successful construction of the P-LAPECS was also evidenced by SEM images (Figure 3D). Here, the GPIFE was fabricated by using polyimide (PI) film as the substrate. Different from the preparation process of GPETFE, the PI film needs to be soaked in dopamine/Tris-HCl (1.5 mg/mL, pH 8.5) solution for 1.0 h before the absorption of gold nanoparticles (AuNPs). The resulting hydrophilic polydopamine layer can significantly enhance the adsorption efficiency of AuNPs seeds on the surface of PI, which is the key step for the seeded growth method. As shown in Figure 3D-a, the conductive gold layer on GPIFE is made up of numerous AuNPs of about ~ 100 nm in size and possesses the advantages of high conductivity (0.9 Ω/sq), excellent flexibility and good biocompatibility for PEC biosensing. Here, after the activation by TCEP, thiolated peptides can be easily modified on the GPIFE through Au-S bonding,


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which makes the surface of the electrode become fuzzy (Figure 3D-b). When the C60/CR-L-cys@SA bioprobe was introduced on the P-LAPECS via the affinity reaction, plenty of granular C60/CR-L-cys are clearly observed (Figure 3D-e). Moreover, the C60/CR-L-cys nanoparticles are obviously reduced when the P-LAPECS was pre-incubated with thrombin (Figure 3D-g), thus revealing the successful construction of the P-LAPECS for thrombin sensing.

Optimization of P-LAPECS. The proper sequence of the substrate peptide is important for the performance of the thrombin sensor. Therefore, we employed two peptides (peptide 1 and peptide 2) which contain both thrombin-cleavable sites and terminal cysteine but with different length to investigate the cleavage efficiency of thrombin. Voltammetric characterizations indicated that the cleavage efficiency of peptide 2 with a shorter length by thrombin was lower than that of peptide 1, reflected by a smaller variation of redox peak current of [Fe(CN)6]3/4- after the cleavage of peptide 2 by thrombin (Figure S7A). The possible reason is that the cleavage site of peptide 2 is too close to the electrode surface, which may hinder the access of thrombin to the cleavage site. In addition, the photocurrent comparison of peptide 1 and peptide 2 for the detection of 1 nM thrombin (Figure S7B) further prove the foregoing result. Therefore, peptide 1 is more suitable for the construction of P-LAPECS for the detection of thrombin. We also examined the effect of cleavage time on the cleavage efficiency of the peptide 1 by 10 nM thrombin. As shown in Figure S7C-D, the photocurrent drops obviously when the incubation time of thrombin varies in the range of 10 ~ 60 min and tends to be stable thereafter, which indicate that the optimal incubation time of thrombin is 60 min. In the case of some other experiment conditions, such as affinity reaction time and temperature, they were referred to a previous literature.



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Detection of thrombin.

Figure 4. Photocurrent responses (A), calibration plot (B) and selectivity evaluation (D) for the detection of thrombin by the P-LAPECS using CHI 830 (n = 3). Part C shows the potential change during the detection of thrombin by the portable pH meter. The concentrations of thrombin on Part A are 0, 0.1, 0.3, 1.0, 3.0, 10, 50, 100, 1000, 10000, 100000 pM (from bottom to top). The concentrations on Part D are 0.1 mg/mL for all the interferents except for thrombin (10 nM) and BSA (1 wt%).

Taking advantage of P-LAPECS for multiplexed biosensing, the sensor was used for the detection of thrombin at a series of concentrations, which enables the establishment of a full calibration plot using just one electrode. From Figure 4A, it is clear that the intensity of photocurrent decreases with increasing the concentration of thrombin, and the photocurrent difference between the background and the target (∆I) is linearly dependent on the logarithm of thrombin concentration (logCThrombin) from 0.1 pM to 1.0 nM, along with a detection limit of 0.05 pM (Figure 4B). Here, the background photocurrent was obtained by directly incubating an uncleaved peptide sensing zone with the bioprobe. Besides the detection of photocurrents by a CHI 830 analyzer, the produced photovoltage responses of P-LAPECS can be also recorded by a handheld pH meter using a two-electrode cell configuration (Figure S3), ACS Paragon Plus Environment

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which is particularly beneficial to the development of portable PEC sensing devices for on-site bioassays.29 Analogously, a linear relationship between the photovoltage difference (∆V) and the logarithm of thrombin concentration (logCThrombin) was obtained in the range of 0.1 pM to 1.0 nM (Figure 4C). The selectivity of the sensor was examined using several model interferents such as rabbit IgG, BSA, lysozyme and trypsin (Figure 4D). Clearly, the sensor shows a small photocurrent change to the interferents at high concentrations when compared to thrombin, suggesting the favorable selectivity of the sensor.

Detection of thrombin inhibitors. As a direct thrombin inhibitor, argatroban can be directly combined with the catalytic site of thrombin to form a steric resistance and inhibit the activity of thrombin.30 While AEBSF, PMSF and Benzamidine are belong to serine protease inhibitor, they can inhibit the activity of both thrombin and some other serine proteases. Here, the potential application of the P-LAPECS for screening thrombin inhibitors was tested using these commercial drugs. As exhibited in Figure 5A, the photocurrent intensity of P-LAPECS increase with the increase of argatroban concentration, indicating argatroban can effectively inhibit the activity of thrombin and lower the cleavage efficiency of thrombin to the substrate peptide. Moreover, the inhibitory curve obtained by the pH meter is close to that acquired by the electrochemical analyzer (Figure 5B), which confirmed the possibility of employing pH meters for PEC screening of thrombin inhibitors. Here, the inhibition percent refers to one minus the photocurrent difference (∆I) ration/photovoltage difference (∆V) ratio of the P-LAPECS incubated with 10 nM inhibited thrombin and 10 nM thrombin. Similarly, the inhibitory curves also displayed the inhibition efficiency of the other three inhibitors (Figure S8A-C).



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Figure 5. (A) Photocurrent responses of P-LAPECS for the detection of thrombin in the presence of argatroban. The concentrations of argatroban are 0, 0.5, 5, 50,500, 5000 and 50000 nM (from top to bottom). (B) Inhibitory efficiency of argatroban. The concentration of thrombin is 10 nM.

The half-maximum inhibition values (IC50), i.e., the concentration of the inhibitor that leads to 50% inhibition of the thrombin activity, for the four inhibitors obtained from the corresponding photoelectrochemical data were estimated to be 1.25 µM (Argatroban), 5.01 mM (AEBSF), 6.31 mM (PMSF) and 3.55 mM (Benzamidine). For different inhibitors, the IC50 values increased with the decreasing inhibition efficiency, which reveal an inhibition efficiency sequence of Argatroban > Benzamidine > AEBSF > PMSF.


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Single P-LAPECS for detecting thrombin and screening its inhibitors Figure 6A shows the results of the activity detection and inhibitor screening of thrombin on single sensor, which indicate that P-LAPECS can provide a comprehensive fingerprint-like detection image and intuitively reflect the inhibiting efficiency difference of different inhibitors (AEBSF, PMSF and Benzamidine) at the same concentration. What’s more, the detection results of the portable pH meter (Figure 6C) are very close to those obtained by the electrochemical analyzer (Figure 6B). These results indicate that the proposed method is easily accessible and can be utilized not only for the detection of thrombin concentration but also for screening its inhibitors. The P-LAPECS also possesses good reproducibility, and a low relative standard deviation (RSD) of 2.99% was obtained for the parallel measurements of 0. 1 nM thrombin by six different sensors (Figure 6D).

Figure 6. Photocurrent responses (A) as well as the corresponding photocurrent (B) and potential change (C) of a single P-LAPECS for the detection of thrombin activity and screening of inhibitors: a. blank; b. 0.1 pM thrombin; c. 10 pM thrombin; d. 10 nM thrombin; e. 10 nM thrombin + 5 µM Argatroban; f. 10 nM thrombin + 5 mM AEBSF; g. 10 nM thrombin + 5 mM PMSF; h. 10 nM thrombin + 5 mM Benzamidine. (D) Photocurrent responses of 0.1 nM thrombin on six different P-LAPECS. ACS Paragon Plus Environment


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Application of the P-LAPECS in human serum. To evaluate the performance of P-LAPECS for real sample analysis by the portable pH meter, the sensor was used for the detection of thrombin in 100-fold diluted human serums. According to the ∆V value obtained in the serum sample (Table 1), the corresponding concentration of thrombin is 0.33 nM, which is in the physiological range of normal thrombin level in blood (lower than nM).31 Furthermore, thrombin with known concentrations was added to two serum samples, and recoveries between 96.2% and 104.1% were obtained. Besides, argatroban (50 µM) added to the serum samples was found to also inhibit the activity (74%) of serum thrombin, which implies that this analysis system may have a promising application for detecting thrombin and its inhibitors in complex bio-samples. Table 1. Thrombin Added (nM)

PEC detection of thrombin in serum samples on P-LAPECS Thrombin found (nM)

Rescovery (%)

RSD (%)

0

0.33

5.2

0.2

0.51

96.2

3.7

0.4

0.76

104.1

2.9

0.6

0.90

96.8

5.3

CONCLUSIONS A portable, self-powered and light-addressable PEC sensor using pH meter detectors for high-throughput drug screening was developed. Using thrombin inhibitors as a drug model, the sensor was fabricated by dividing the surface of a gold film electrode into eight separated zones and identically constructing biotin-labeled and thrombin-cleavable peptide sensing arrays on each zone, which together with a streptavidin-labeled C60/CR-L-cys bioprobe enables the sensitive detection of thrombin at different concentrations or screening of its multiple inhibitors using one electrode. Beneficial from the high conversion efficiency of C60/CR-L-cys, the P-LAPECS can detect thrombin in the wide calibration ACS Paragon Plus Environment

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range of 0.1 pM ~ 1.0 nM and with a low detection limit of 0.05 pM using a handheld pH meter, which is successfully applied to the detection of thrombin and evaluation of inhibitor activity in serum samples. As far as we know, this is the first example that demonstrates the potential application of portable pH meters for multiplexed PEC biosensing, which opens a new door for the development of portable and highly integrated PEC analytical devices.

ACKNOWLEDGMENTS. We thank the financial support of the National Nature Science Foundation of China (No. 21675116), the National Natural Science Foundation of China (No. 21775050), the National Basic Research Program of China (973 Program, No. 2015CB352100) and the Fundamental Research Funds for the Central Universities (No. 2042014kf0244).

Supporting Information Available. Additional text describing detail experimental procedures, 8 figures showing schematic procedures for the preparation of C60/CR-L-cys@SA bioprobe and the construction of the P-LAPECS, the photos of the detection mode of P-LAPECS, SEM images of materials, P-LAPECS applied to detection of rabbit IgG, optimization of conditions, inhibitory efficiency of AEBSF, PMSF and benzamidine. This material is available free of charge via the Internet at http://pubs.acs.org.

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Portable, Self-Powered and Light-Addressable Photoelectrochemical

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