Installing Logic Gates in Permeability Controllable Polyelectrolyte

Jul 31, 2015 - Proteases and nucleases are enzymes heavily involved in many important biological processes, such as cancer initiation, progression, an...
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Installing Logic Gates in Permeability Controllable PolyelectrolyteCarbon Nitride Films for Detecting Proteases and Nucleases Lichan Chen,†,§ Xiaoting Zeng,† Anirban Dandapat,§ Yuwu Chi,*,† and Donghwan Kim*,§,‡ †

MOE Key Laboratory of Analysis and Detection Technology for Food Safety, State Key Laboratory of Photocatalysis on Energy and Environment, and College of Chemistry, Fuzhou University, Fuzhou, Fujian 350108, China § School of Chemical and Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive, Singapore 637457, Singapore ‡ School of Chemical Engineering, Sungkyunkwan University, 16419, Republic of Korea S Supporting Information *

ABSTRACT: Proteases and nucleases are enzymes heavily involved in many important biological processes, such as cancer initiation, progression, and metastasis; hence, they are indicative of potential diagnostic biomarkers. Here, we demonstrate a new label free and sensitive electrochemiluminescent (ECL) sensing strategy for protease and nuclease assays that utilize target-triggered desorption of programmable polyelectrolyte films assembled on graphite-like carbon nitride (g-C3N4) film to regulate the diffusion flux of a coreactant. Furthermore, we have built Boolean logic gates OR and AND into the polyelectrolyte films, capable of simultaneously sensing proteases and nucleases in a complicated system by breaking it into simple functions. The developed intelligent permeability controlled enzyme sensor may prove valuable in future medical diagnostics.

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Recently, we have found that g-C3N4 nanomaterials harbor strong ECL activity, and the sensors taking advantage of excellent film-forming ability and ECL stability of gold nanoparticle-g-C3N4 nanosheet nanohybrid (Au-g-C3N4) may eliminate the complicated conjugation process and perform in a convenient way for biosensing.19−21 It is well-established that a layer-by-layer (LBL) self-assembly technique22 allows programmable buildup of a hybrid polyelectrolyte multilayered (PEM) film with diverse charged functional materials.23 This advantage enables the LBL technique to design enzymatic biodegradable multilayered capsules for drug delivery via suitably selecting of the polyelectrolyte constituents.24−29 In these LBL-capsules, sustain release of the encapsulated drugs can be realized.24−29 Inspired by these works, we present here a sensitive and convenient ECL sensing strategy for assays of protease and nuclease based on target-trigged decomposition of the PEM assembly film. As illustrated in Scheme 1, desorption of polyelectrolyte films triggered by enzyme hydrolysis of polyelectrolyte decreases the thickness of the assembly film, enlarges the flux of coreactant, and thus induces the recovery of the ECL intensity of Au-g-C3N4 ECL film that was inhibited by the deposition of the assembly film. Proper LBL assembly with different substrates enables the sensor to respond to protease and nuclease, respectively. Programmable OR and AND

ssays for proteases and nucleases are of high importance in many normal biological processes as well as in diseases, such as cancer, stroke, and infection, due to their special functions.1−3 The current biosensors to assay protease and nucleases are mainly built on the fluorescence resonance energy transfer (FRET) 4−6 strategy using fluorescently tagged substrates and colorimetric approaches2,7,8 taking advantage of the color change resulting from the enzyme-induced aggregation or dissociation of gold nanoparticles. Although fluorescent labels can provide high sensitivity, chemical modification may reduce the catalytic efficiency of the enzymes and increase analysis time and assay complexity. Colorimetric assays are convenient to use but show relatively low sensitivity. Therefore, development of convenient methods with high sensitivity for proteases and nucleases is still in great demand. Electrochemiluminescence (ECL), a light emission process triggered by electrochemical methods via redox reactions of electrogenerated reactants, is a powerful technique for developing sensitive and convenient biosensors due to its unique features including high sensitivity, versatility, and controllability.9,10 Therefore, the introduction of ECL sensing technology into the assays of proteases and nucleases may be of considerable research interest and clinic significance. Graphite-like carbon nitride (g-C3N4), representing an important class of conjugated polymer semiconductors, has been widely used in photocatalysis of water splitting,11−14 bioimaging,15 and sensing16−18 in the past few decades, due to its catalytic, fluorescent, and biocompatible properties. © XXXX American Chemical Society

Received: May 22, 2015 Accepted: July 31, 2015

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DOI: 10.1021/acs.analchem.5b01916 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

procedure was then carried out for the next polyelectrolyte. The prepared electrodes were stored in 10 mM PBS (pH 7.4) in the dark prior to enzyme reaction. The different polyelectrolyte solutions were prepared as follows: (1) PLL and PAA at 2 mg mL−1 in 20 mM HEPEs buffer (pH 7.4) with 75 mM NaCl, respectively; (2) PEI and DNA at 0.2 mg mL−1 in 10 mM PBS (pH 7.4), respectively. Enzyme Reaction. The electrodes modified with different PEM films were soaked in the enzyme reaction solution at 37 °C, respectively, followed by rinsed throughout with water and kept in 10 mM PBS (pH 7.4) prior to ECL experiments. The enzyme reaction solutions for different PEM films are prepared as follows: (1) trypsin detection using (PLL/PAA)m film: trypsin solution of various concentrations in 10 mM PBS buffer (pH 7.4); (2) DNase I detection using (PEI/DNA)nPEI film: DNase I solution of various concentrations in 10 mM Tris-HCl buffer (pH 7.6) containing 2.5 mM MgCl2 and 0.5 mM CaCl2; (3) “OR” and “AND” logic gate: both trypsin and DNase I in 10 mM Tris-HCl buffer (pH 7.6) containing 2.5 mM MgCl2 and 0.5 mM CaCl2. ECL and CV Detection. The ECL responses of the sensors before and after treatment with enzyme were recorded in 0.1 M PBS (pH 7.4) containing 10 mM K2S2O8, with a potential range of 0 to −1.1 V at a scan rate of 100 mV s−1. Cyclic voltammetry (CV) was carried out in 0.1 M, pH 7.4 PBS containing 10 mM K4[Fe(CN)6], with a potential range of 0 to 0.5 V at a scan rate of 100 mV s−1. Both ECL and CV measurements were performed on an ECL detection system (MPI-E, Remex Electronic Instrument Lt. Co., Xi’an, China) with a conventional three-system composed of a modified GCE working electrode, a Pt wire counter electrode, and a Ag/AgCl (3 M KCl) reference electrode. SEM and AFM Samples Preparation. Silica substrates were pretreated using Piranha solution (3:1 of volume ratio of H2SO4 vs HCl). The functionalized silica substrates were applied to LBL assembly of polyelectrolyte films using the aforementioned process for electrode modification. All samples were thoroughly rinsed with doubly distilled water and dried under a N2 stream before characterization. The capture of field emission scanning electron microscope (FESEM) images was conducted on a Nova NanoSEM 230 field-emission microscope (FEI, USA). The heights of the PEM films were obtained from an atomic force microscope (AFM) (Nanoman, Veeco, Santa Barbara, CA) using tapping mode. The root-mean-square (RMS) roughness values were calculated by the software.

Scheme 1. Schematic Representation of Label-Free ECL Protease and the Nuclease Assay by Turning on ECL Emission of Au-g-C3N4 Film upon Desorption of Polyelectrolyte Film To Improve the Flux of Coreactant S2O82−

Boolean logic-gate allows the sensor to simultaneously assay proteases and nucleases in one sample. To the best of our knowledge, no material other than DNA30,31 has been used for the construction of the logic-based ECL sensing platform. Furthermore, for this ECL sensing strategy, no label is required on the substrates because of the electrostatic assembly; high sensitivity could be achieved by (i) the turn-on assay that shows higher sensitivity and a lower chance of a false positive signal as compared to the turn-off assay; (ii) preconcentration of targets via proper selection of the outermost layer of PEM film.

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EXPERIMENTAL SECTION Materials. Dicyanamide, hydrogen tetrachloroaurate trihydrate (HAuCl4·3H2O), poly-L-lysine (PLL, Mw ∼ 70 000− 150 000), and trypsin (1000 units mg−1) were purchased from Sigma. Poly(acrylic acid) (PAA, Mw ∼ 240 000) was ordered from Alfa Aesar. Poly(ethylenimine) (PEI, Mw ∼ 10 000) and N-[2-hydroxyethyl] piperazine-N′-[2-ethanesulfonic acid] (HEPEs, free acid, high purity grade) were obtained from Aladdin Chemistry Co., Ltd. (Shanghai, China). DNA (sequence: 5′-CCG GTG GGT GGT CAG GTG GGA TAG CGT TCC GCG TAT GGC CCA GCG CAT CAC GGG TTC GCA CCA-3′) was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). DNase I (RNase free, 2000 units) was ordered from Biolabs. Potassium persulfate (K2S2O8), sodium borohydride (NaBH4, 96.0%), and sodium citrate were ordered from Fuchen Chemical Reagent Co. (Tianjin, China). All of the other chemicals were of reagent grade and used as received. Doubly distilled water was used throughout this work. LBL Assembly of Polyelectrolytes on Au-g-C3N4/GCE. Prior to each step, the glassy carbon electrode (GCE, 3 mm in diameter) was polished using 0.3 and 0.05 μm of alumina slurry in succession and washed thoroughly with water using ultrasonic cleaning. Then, 3 μL of the prepared gold nanoparticle-graphite-like carbon nitride nanosheet nanohybrid (Au-g-C3N4) suspension, which was prepared from AuCl4- and g-C3N4,20 was coated onto the precleaned GCE and dried at 25 °C for over 3 h. The modified electrodes were washed with 10 mM PBS (pH 7.4) to remove unabsorbed materials. For LBL assembly, the modified electrodes were first immersed into a positively charged polyelectrolyte solution for 30 min followed by two rinsing steps (5 min each) in wash beakers. This

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RESULTS AND DISCUSSION Trypsin Assays Using PLL/PAA PEM Film. Mass transport through the PEM film is purely diffusive. For a given PEM film, the steady-state diffusion-limited current (im) and flux (Jm) of electroactive species through the PEM thin film is highly dependent on the multilayer thickness (d).32,33 Jm =

im DC ̅ ̅ = nFA d

(1)

where D̅ , C̅ , n, F, and A refer to the membrane diffusion coefficient, the concentration of the electroactive species, electron transfer number, Faraday’s constant, and the electrode area, respectively. eq 1 indicates that both deposition and desorption of PEM film can be easily monitored by the emission intensity change of solid-state ECL film due to the high dependence of diffusion flux of coreactant on the PEM B

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Analytical Chemistry film thickness as well as the high dependence of the emission of ECL film on the diffusion flux of coreactant. For proof of principle, trypsin was selected as the representative protease. As a common reagent in cell biology, trypsin plays a pivotal role in regulating the pancreatic exocrine function and specially cleaves the PLL chains at the carboxyl side of the amino acids lysine.34−36 As illustrated in Figure 1A,

g‐C3N4 + e− → g‐C3N4•−

(2)

S2 O82 − + e− → SO4 2 − + SO4•−

(3)

SO4•− + g‐C3N4•− → g‐C3N4* + SO4 2 −

(4)

g‐C3N4* → g‐C3N4 + hν

(5)

PLL and PAA were alternately assembled on the negatively charged Au-g-C3N4 film to construct the enzymatic biodegradable PEM film. We preferred to have polyanion PAA as the outermost layer as the negative surface charge should condense trypsin, considering that trypsin was cationically charged in PBS buffer at pH 7.4 (the isoelectric point of trypsin = 8.75). The electrostatic assembly procedure allows for eliminating the requirement for chemical modification of the substrate. The layer buildup on Au-g-C3N4 film was monitored by the ECL intensity change of Au-g-C3N4 film, taking advantage of the dependence of the S2O82− flux on the PEM film thickness. Figure S1 shows the progression of ECL as more bilayers of PLL/PAA are assembled: the ECL intensity decreases sharply and then plateaus after seven bilayers. The ECL intensity of the Au-g-C3N4 film upon deposition of seven bilayers of PLL/PAA (denoted as (PLL/PAA)7 where the subscript refers to number of bilayers) is about 16 times lower than that of Au-g-C3N4 film. The small decline in ECL intensity could be ascribed to the low blocking efficiency of PLL/PAA PEM film toward S2O82− transport, which results from the exponential growth regime of the film. It is well-known that, when PAA is introduced to polypeptides such as the PLL layer, the PLL/PAA complex formed and reorganized into small islands; as LBL buildup continued, PLL chains diffuse in and out through the films and eventually afford exponentially growing film with loosely packed and porous structure and island-like domains and a rough surface.36−38 Then, we optimized the number of bilayers of the PEM film for the trypsin assay to be seven (Figure S2), since (PLL/PAA)7 film presents the largest ECL enhancement factor (I − I0)/I0 (where I0 and I refer to the ECL intensity of the (PLL/PAA)7/Au-g-C3N4 film before and after degradation with trypsin, respectively). The buildup of the (PLL/PAA)7 film on the Au-g-C3N4 film was confirmed by SEM. Figure 1 shows that molecularly smooth PEM film (Figure 1C) was formed on Au-g-C3N4 film (Figure 1B) after (PLL/PAA)7 deposition.39 Furthermore, lots of island-like domains can be dimly observed on bare silicon substrate, which is a feature of exponential growth of the PLL/PAA film. The detail surface topographic feature of the PEM film was further studied by AFM. As shown in Figure 1E and Figure S3A, the (PLL/PAA)7 film (thickness of about 6 nm) interspersed with randomshaped island-like domains (average height of about 40 nm) was clearly observed, verifying an exponential growth of films. The root-mean-square (RMS) roughness was measured to be 10.9 nm. Subsequently, we evaluated the ECL sensor with (PLL/ PAA)7/Au-g-C3N4 film for trypsin assays. The stability of the sensor was first investigated by incubating the modified electrode at 37 °C in trypsin-free enzyme reaction solution for 80 min. The sensor maintains a low and constant response (curve with blue squares in the inset I of Figure 1G), indicating the excellent stability. In the presence of trypsin, the hydrolysis of the PLL-induced chain effect, including the gradual desorption of the PEM film, the increase of diffusion flux of S2O82−, and thus the recovery of ECL intensity of the Au-g-

Figure 1. Trypsin assays based on target-triggered degradation of PLL in the (PLL/PAA)7 assembly film. (A) Schematic representation. SEM images in isometric view (60° from normal) of the edge of Au-g-C3N4 film (B), (PLL/PAA)7 film on Au-g-C3N4 film (C), and the sensing interface after incubation in 10 mM PBS, pH 7.4, containing 50 U mL−1 trypsin at 37 °C for 60 min (D). All the samples were coated with 5 nm Pt. 3D AFM images of (PLL/PAA)7 film degraded without (E) and with (F) 50 U mL−1 trypsin at 37 °C for 60 min. The scale of area was 2 × 2 μm. (G) ECL responses of (a) Au-g-C3N4 film, (b) (PLL/PAA)7/Au-g-C3N4 film, and (c) (b) upon degradation with 50 U mL−1 trypsin for 60 min. The inset I in G shows ECL response of (PLL/PAA)7/Au-g-C3N4 film as a function of degradation time in 10 mM PBS, pH 7.4, without (blue squares) and with (red circles) 50 U mL−1 trypsin. (H) Dose responses of ECL enhancement factor vs trypsin concentration: (a) 0, (b) 0.05, (c) 0.5, (d) 5, (e) 50, and (f) 500 U mL−1. Error bars were derived from three parallel assays. The established assay is a two-step sensing method in which the sensor is first incubated in enzyme reaction solution and then analyzed in ECL test solution.

Au-g-C3N4 film was first anchored on GCE as solid state ECL film due to its superior ECL efficiency and stability as well as strong S2O82− concentration-dependent ECL emission as earlier reported by us.19−21 The ECL emission could be recorded during cathodic polarization due to the following ECL reactions:19 C

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Analytical Chemistry C3N4 film, was expected. Indeed, as shown in the inset II in Figure 1G, the ECL intensity increases as a function of degradation time in trypsin (50 U mL −1), and ECL enhancement of 2.2 times was obtained after incubation for 60 min (curve c in Figure 1G). The gradual increase in ECL intensity confirmed the progressive desorption of PEM film. CV provides a further assessment of the change in ion diffusion pathways upon deposition and desorption of PEM film, in which higher peak currents and smaller peak potential separation suggest better access to the electrode.40 As shown in Figure S4, a broad and plateau-shaped CV (curve b) resulted from PEM film deposition, turning into a quasi-reversible CV along with larger peak currents (curve c) as the sensor was soaked in 50 U mL−1 trypsin solution for 60 min, suggesting the enlargement of the flux of Fe(CN)64− upon the desorption of PEM film. SEM and AFM were used to investigate the structure change of the PEM films upon degradation with 50 U mL−1 trypsin. Both the molecularly smooth PEM film on Au-gC3N4 and the island-like domains on the bare silicon substrate became less obvious (Figure 1D). As shown in the AFM image in Figure 1F and Figure S3B, the thin (PLL/PAA)7 film almost disappeared and the configurations of the islands became blunt and short; the RMS roughness decreased to around 4.3 nm. According to the previous report,14 the RMS roughness change of the PEM film with an exponentially growing regime coincides with the change in film thickness during the process of film deposition and desorption. Therefore, the RMS roughness change was used in the subsequent experiments. Next, we investigated the performance of the ECL sensor for trypsin assays. As shown in Figure 1G, the ECL sensor can be used for the quantitative detection of trypsin in the concentration range of 0.02 to 500 U mL−1 with a limit of detection of 0.01 U mL−1 (three times the standard deviation of the background noise). The high detection sensitivity may be attributed to (i) trypsin being electrostatically condensed onto the PAA surface; (ii) Au-g-C3N4 film revealing a strong S2O82− concentration-dependent ECL emission. The analytical performance of the ECL sensor for the trypsin assay was compared with that of other methods (Table S1). The comparison suggests that the ECL sensor is one of the excellent sensors for the trypsin assay. Deoxyribonuclease I Assays Using PEI/DNA PEM Film. To address the generality of the proposed approach, we explore another PEM film containing DNA for the detection of nuclease. Deoxyribonuclease (DNase) I was chosen as the representative nuclease since it plays a key role in biological processes and can act as a biomarker for lupus erythematosus diagnosis.7,41 DNase I digests DNA at phosphodiester linkages in the presence of Mg2+ and Ca2+ ions. During digestion, both Mg2+ and Ca2+ ions coordinate to the phosphodiester of DNA; Ca2+ ions stabilize the structure of DNase I.24 The alternate LBL self-assembly of positively charged polyethylenimine (PEI) and negatively charged DNA were explored on Au-g-C3N4 film to construct the DNase I biodegradable PEM film (Figure 2A). The outermost layer of PEI was used for electrostatic condensation of DNase I, which was anionically charged in PBS solution at pH 7.4 (the isoelectric point of DNase I = 5.0). The ECL intensity of Au-g-C3N4 film was sharply inhibited by (PEI/DNA)1PEI deposition and further reduced as more bilayers of adsorption (Figure S5). We optimized the number of bilayers of the PEM film for the DNase I assay to be five based on ECL enhancement factor (Figure S6). The ECL intensity of (PEI/DNA)5PEI/Au-g-C3N4 film (curve b in

Figure 2. DNase I assays based on target-triggered degradation of DNA in the (PEI/DNA)5PEI assembly film. (A) Schematic illustration. (B) ECL responses of (a) Au-g-C3N4 film, (b) (PEI/ DNA)5PEI/Au-g-C3N4 film, and (c) (b) upon degradation with 20 U mL−1 DNase I for 40 min. The inset I in B shows ECL response of (PEI/DNA)5PEI/Au-g-C3N4 film as a function of degradation time in 10 mM Tris-HCl, pH 7.6, containing 2.5 mM MgCl2 and 0.5 mM CaCl2 without (blue squares) and with (red circles) 20 U mL−1 DNase I. (C) Dose responses of ECL enhancement factor vs DNase I concentration: (a) 0, (b) 0.05, (c) 0.5, (d) 5, (e) 50, and (f) 200 U mL−1. Error bars were derived from three parallel assays.

Figure 2B) is about 80 times lower than the that of Au-g-C3N4 film (curve a in Figure 2B). This can be explained by the lower permeability efficiency of the (PEI/DNA)5PEI film toward S2O82− that results from the linear growing regime of the PEM film with densely packed structure. The AFM micrograph along the edge of a scratch made in the (PEI/DNA)5PEI film (Figure S7) presents the relatively smooth and ordered surface of the film, verifying the linear growth regime. Subsequently, we evaluated the sensing performance of (PEI/DNA)5PEI/Au-gC3N4 film for DNase I assays. The stability of the sensor was first investigated by incubating the modified electrode at 37 °C in DNase I-free enzymatic reaction solution for 60 min. The ECL intensity of the electrode shows a slight increase as a function of immersion time (curve with blue squares in the inset I of Figure 2B). The slight increase of ECL intensity is presumably because coordination of the Mg2+ and Ca2+ to the DNA alters the intrinsic charge balance between DNA and PEI layers, thus leading to a change in the desorption of (PEI/ DNA)5PEI film in the presence of 20 U mL−1 DNase I. ECL enhancement of 2.7 times was obtained after incubation for 40 min (curve c in Figure 2B). The proposed ECL sensor can be used for linear detection of DNase I in the concentration range of 0.008 to 200 U mL−1 with a limit of detection of 0.008 U mL−1 (Figure 2C). The comparison of ECL sensor with other sensors indicates that the ECL sensor has good sensing performance for the DNase I assay (Table S1). Sensing Performance of the PEM Films in the Human Urine Samples. To evaluate their applicability, the proposed ECL sensors were applied for trypsin and DNase I assay in human urine samples. We first demonstrated the robustness of the sensor against possible interferences present in urine. As shown in Figure S8, 50 U mL−1 of trypsin and DNase I induced large ECL enhancement of (PLL/PAA)7 and (PEI/DNA)5 PEM films, respectively, while 50 U mL−1 of thrombin, pepsin, lysozyme, and GOx resulted in only slight ECL enhancement of D

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Analytical Chemistry both PEM films. The observed high selectivity indicates that the proposed ECL sensors have potential in the urine sample assay. Then, the sensing performance for clinical diagnosis was carried out by directly measuring trypsin and DNase I, as well as adding a fixed amount of them, in urine samples taken from two healthy volunteers. The ECL enhancement of the diluted urine samples show detectable trypsin and DNase I, as shown in Table S2. Both of the recoveries of the supplemented trypsin and DNase I were within 100 ± 10%, indicating the potential of the developed permeability controlled enzyme sensors for clinical diagnostics. Logic-Gate Response by Programmable Assembly of PEM Film. Boolean logic operation that breaking a complex system into simple functions enables us to detect the targets in a predictable way.42 We sought to construct functional PEM films that exhibited a Boolean logic-gate response toward tryspin and DNase I as inputs via appropriate polyelectrolyte selection. We first designed the OR gate as depicted in Figure 3A. The PEM film used is simply fabricated by LBL assembly of

DNA being much lower than that of PAA. In the input state of (1,0), i.e, I1 = 1 and I2 = 0, PLL digestion decomposed the PEM film and reduced the RMS roughness to 0.38 nm (Figure 3C), resulting in ECL enhancement of 1.86 times (Figure 3F). Similarly, in the input state of (0,1), DNA degradation also decomposed the PEM film and reduced the RMS roughness to 0.63 nm (Figure 3D), leading to ECL enhancement of 1.94 times (Figure 3F). In the input state of (1,1), both enzymatic degradation of PLL and DNA decomposed the PEM film more thoroughly and reduced the RMS roughness to 0.57 nm (Figure 3E), giving rise to ECL enhancement of 3.37 times (Figure 3F). The “sharp spines” in Figure 3D,E may be ascribed to the absorption of DNase I on the remainder of the PEM film. Furthermore, application of the (0,0) input did not obviously change in ECL intensity (curve (0,0) in Figure 3F). CV was used for further assessment of the change in ion diffusion pathways under different combinations of the input. The overall trends of current and peak potential variation (Figure S11) are in good agreement with the change of ECL emission (Figure 3F). By defining that the enhancement factor above the threshold of 0.5 has an output value of “1”, whereas the enhancement factor below 0.5 corresponds to an output value of “0”, we may find that the features of the system agree with an OR logic circuit (Figure 3F−H). Namely, either one of the inputs caused an ECL intensity increase as the output. We further designed the AND gate via appropriate assembly of polyelectrolytes. When the advantages of programmable LBL assembly, gradual desorption of PEM film by enzyme digestion, and the specificity of the enzyme were utilized, the composition of the PEM film for AND gate fabrication was designed to be PLL/PAA/PLL/DNA/PEI/DNA/PLL/PAA/PLL/DNA (Figure 4A). ECL was also used to monitor the fabrication process (Figure S12). The ECL intensity of Au-g-C3N4 film was inhibited by 240 times upon the PEM film deposition. The effective blockage of S 2O 82− may be ascribed to the

Figure 3. OR logic gate design and implementation. (A) Schematic illustration. 3D AFM images of (PLL/DNA)7 film degraded (B) without, (C) with input (1,0), (D) with input (0,1), and (E) with input (1,1) at 37 °C for 60 min. The scale of the area was 2 × 2 μm. (F) ECL responses before (dashed line) and after the input signals (0,0), (1,0), (0,1), and (1,1). (G) Bar diagram derived from (F) showing the ECL enhancement factor for different input signals. The dashed line shows the threshold (0.5). (H) Circuit and truth table for an OR logic gate. Input 1: 50 U mL−1 tryspin. Input 2: 50 U mL−1 DNase I.

positively charged tryspin-degradable PLL and negatively charged DNase I-degradable DNA. In order to perform the logic operations, both the absence of tryspin (Input 1, I1) and DNase I (Input 2, I2) were considered as input value “0”, whereas both the presence of tryspin (50 U mL−1) and DNase I (50 U mL−1) were considered as input value “1”. The PLL/ DNA PEM assembly process was monitored by ECL (Figure S9), and the number of bilayers of the PLL/DNA film were optimized to be seven (Figure S10). The ECL intensity of (PLL/DNA)7/Au-g-C3N4 film was 10 times lower than that of Au-g-C3N4 film, resulting from the exponential growth regime of the PEM film that was confirmed by the island-like domains and rough surface (RMS roughness of 0.72 nm) shown in AFM (Figure 3B). The size of the island-like domains of PLL/DNA film (Figure 3B) is obviously smaller than that of the PLL/PAA film (Figure 1E), which can be due to the molecular weight of

Figure 4. AND logic gate design and implementation. (A) Schematic illustration. 3D AFM images of PLL/PAA/PLL/DNA/PEI/DNA/ PLL/PAA/PLL/DNA film degraded (B) without, (C) with input 1, (D) with input 2, and (E) with input 1 and 2 at 37 °C for 60 min. The scale of area was 2 × 2 μm. (F) ECL responses before (dashed line) and after the input signals (0,0), (0,1), (1,0), and (1,1). (G) Bar diagram derived from (F) showing the ECL enhancement factor for different input signals. The dashed line shows the threshold (1.5). (H) Circuit and truth table for an AND logic gate. Input 1: 50 U mL−1 tryspin. Input 2: 50 U mL−1 DNase I. E

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Analytical Chemistry combination of exponential and linear growing regime of the PEM film. The diffusion of PEI/DNA into the previously formed PLL/PAA/PLL/DNA porous structure may result in the densely packed structure. Treatment of the sensor with input (1,0) resulted in the degradation of PLL in the outer layers of the film, while keeping the inter layers unaffected, as depicted in Figure 4A. In this process, the RMS roughness of the film was reduced from 3.09 nm (Figure 4B) to 2.25 nm (Figure 4C), leading to an enhancement factor of 0.83 (curve (1,0) in Figure 4F). On the other hand, treatment of the sensor with input (0,1) leads to the digestion of DNA on the outermost layer of the film, while retaining the other layers unperturbed. This was confirmed by AFM (Figure 4D) that the RMS roughness of the film was only reduced by 0.3 nm. As a result, no measurable ECL signal change was detected (curve (0,1) in Figure 4F). However, treatment of the sensor with input (1,1) resulted in drastically reducing the RMS roughness from 3.09 to 1.62 nm (Figure 4E) and leads to an ECL enhancement factor of 2.77 (curve (1,1) in Figure 1F). The corresponding ECL enhancement factor bar diagrams (Figure 4G) exhibit that only the (1,1) input leads to the output value of “1”. The CV result (Figure S13) corresponds to the change of ECL emission (Figure 4F). By defining enhancement factor of 1.5 as the threshold, we may find that the features of the system concur with an AND logic circuit (Figure 4F−H). Namely, the gate output is activated only when the both inputs (i.e., (1,1)) are present.43

ACKNOWLEDGMENTS

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CONCLUSIONS By combining target-triggered gradual decomposition of functional PEM film, thickness-dependent diffusion flux of coreactant through the film, and coreactant concentrationdependent ECL intensity of the ECL film, we have successfully developed signal-on solid-state ECL sensors for protease and nuclease assays. The ECL sensors can be easily fabricated via the low-cost and label-free LBL assembly technique and show high sensitivity toward protease and nuclease detection taking advantage of target preconcentration on the surface of the PEM film as well as strong S2O82− concentration dependent Au-gC3N4 ECL emission. More interestingly, the PEM film that contains programmable substrates showed a capacity for OR or AND logic-type sensing toward protease and nuclease as input stimuli. The proposed sensor can be expected to be a suitable sensing platform for medical diagnostics and high-throughput drug screening. ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b01916.

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This study was financially supported by National Natural Science Foundation of China (21375020) and Specialized Research Fund for the Doctoral Program of Higher Education of China (20133514110001). D.H.K. acknowledges the financial supports of the Ministry of Education of Singapore (MOE2012-T2-1-058), and the Ministry of Science, ICT & Future Planning (NRF-2013M3C8A3078512).

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Article

Figures S1−S13, and Tables S1−S2 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Fax: +86-591-22866137. Tel: +86-591-22866137. E-mail: y.w. [email protected]. *E-mail: [email protected]; [email protected]. Notes

The authors declare no competing financial interest. F

DOI: 10.1021/acs.analchem.5b01916 Anal. Chem. XXXX, XXX, XXX−XXX

Article

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DOI: 10.1021/acs.analchem.5b01916 Anal. Chem. XXXX, XXX, XXX−XXX