Ultrasensitive Photoelectrochemical Biosensing of Multiple

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Ultrasensitive Photoelectrochemical Biosensing of Multiple Biomarkers on a Single Electrode by a Light Addressing Strategy Juan Wang, Zhihong Liu, Chengguo Hu, and Shengshui Hu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02148 • Publication Date (Web): 20 Aug 2015 Downloaded from http://pubs.acs.org on August 26, 2015

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Analytical Chemistry

Ultrasensitive Photoelectrochemical Biosensing of Multiple Biomarkers on a Single Electrode by a Light Addressing Strategy Juan Wang, Zhihong Liu, Chengguo Hu,* Shengshui Hu 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 to: Dr. Chengguo Hu, College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China; Fax: +86-27-68754067; Email: [email protected].

ABSTRACT: Ultrasensitive multiplexed detection of biomarkers on a single electrode is usually a great challenge for electrochemical sensors. Here, a light addressable photoelectrochemical sensor (LAPECS) for the sensitive detection of multiple DNA biomarkers on a single electrode was reported. The sensor was constructed through four steps: (1) immobilization of capture DNA (C-DNA) of different targets on different areas of a single large-sized gold film electrode; (2) recognition of each target DNA (T-DNA) and the corresponding biotin-labeled probe DNA (P-DNA) through hybridization; (3) reaction of the biotin-labeled probe DNA with a streptavidin-labeled all-carbon PEC bioprobe; (4) PEC detection of multiple DNA targets one by one via a light addressing strategy. Through this principle, the LAPECS can achieve ultrasensitive detection of three DNA sequences related to hepatitis B (HBV), hepatitis C (HCV) and human immunodeficiency (HIV) viruses with a similar wide calibration range of 1.0 pM ~ 0.1 µM and a low detection limit of 0.7 pM by using one kind of PEC bioprobe. Moreover, the detection throughput of LAPECS may be conveniently expanded by simply enlarging the size of the substrate electrode or reducing the size of the sensing arrays and the light beam. The present work thus demonstrates the promising applications of LAPECS in developing portable, sensitive, high-throughput and cost-effective biosensing systems. 1

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KEYWORDS: Photoelectrochemical biosensors; light addressable; multiplexed detection; fullerene; DNA

INTRODUCTION Electrochemical sensing techniques have been increasingly popular for their advantages in terms of speed, expense, sensitivity and integration.1 Due to the synchronous electronic response nature of conductive substrates, it is difficult to carry out multiplexed electrochemical detection using different areas of a single electrode. Currently, two approaches are often used to overcome the detection throughput of electrochemical sensors. The first approach involves the employment of electrode arrays and multi-channel analyzers,2-4 and the other one relies on the development of multiple redox tags.5,6 However, these approaches usually suffer from complicated fabrication of electrode arrays and high cost of multi-channel instruments or limitations of available redox probes and potential windows. Photoelectrochemical (PEC) sensors are a kind of analytical devices that employ common electrochemical analyzers and additional light sources but rely on photo-induced electron transfer (PET) processes.7 Apart from the advantages resembling electrochemical sensors, PEC sensors also possess the merit of high sensitivity due to the difference of excitation and response signals.8-10 Theoretically, it is easier for PEC sensors to realize high-throughput detection as compared with electrochemical sensors. The reasons include: (i) the high spatial resolution of excitation light sources allows the detection of multiple samples in different areas of a single electrode through a light addressing strategy; (ii) PEC sensing at open circuit potentials enables the employment of large-sized substrate electrodes but with low backgrounds; (iii) the on/off excitation of light sources offers a chance for the calibration of the base line for each sensing area. Unfortunately, most of previous works on PEC sensors mainly focus on the development of sensing platforms with high sensitivity or selectivity for a wide range of individual analytes, e.g., metal ions,11,12 toxicants,13,14 biomolecules,15-17 cells,18,19 and so on. Only a few works establish PEC sensors for multiple targets by using electrode arrays20-22 or multi-channel electrochemical

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analyzers.23 The light-addressable potentiometric sensor (LAPS) is a semiconductor-based chemical sensor with an electrolyte/metal-insulator-semiconductor (EIS/MIS) structure, on which electron-hole pairs are generated at the irradiated position of the semiconductor layer to obtain a photocurrent with exerted bias potentials.24 The amplitude of the light-induced photocurrent of LAPS is sensitive to the surface potential related to electrochemical events on the insulator layer,25 and therefore LAPS is a powerful label-free tool to detect multiple targets like heavy metals,26 solution pH27 and circulating tumor cells28 by light addressing excitation. However, special instruments such as frequency-modulated light sources, lock-in amplifiers and demodulation of the collected photocurrent responses29,30 are usually required to differentiate the signals of different targets by this technique, which is unfavorable to the development of portable and cost-effective analytical devices. Here, a light-addressable photoelectrochemical sensor (LAPECS) for the multiplexed detection of biomarkers on a single electrode was reported. This single-electrode light addressing strategy has several very attractive properties: (i) simple construction of substrate electrodes; (ii) employment of singlechannel electronic analyzers with simple instrument and facile operation; (iii) possibility of constructing self-calibration and self-driving PEC biosensing systems. For the purpose of multiplexed biosensing, a large-sized gold-polyethylene terephthalate (PET) composite film electrode (GPETFE) (Figure S1), which was fabricated by a simple electroless method and possessed excellent conductivity, flexibility and size controllability,31,32 was employed as the substrate electrode. By coupling a sandwiched DNA sensing structure with an affinity-based signal transduction system, an all-carbon photovoltaic material, i.e., the carboxylated multiwalled carbon nanotube-Congo red-fullerene nanohybrid (MWNTCOOHCR-C60),33 was introduced onto different sites of a single GPETFE for different DNA targets (Figure 1). Through a simple light addressing strategy, the LAPECS was able to detect three DNA sequences related to HBV, HCV and HIV viruses with high sensitivity and selectivity. Although the light addressing strategy used here is similar to the well-developed LAPS, the LAPECS works in a more

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direct manner that the sensor can be constructed on common substrate electrodes and may employ inexpensive current detection devices like electrochemical analyzers, galvanometers or even glucose meters, which should be particularly suitable for developing portable, sensitive, high-throughput and inexpensive biosensing systems.

Figure 1. Schematic representation for the multiplexed DNA detection at a single LAPECS using an allcarbon PEC bioprobe.

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), sodium chloride (NaCl), hydrochloric acid (HCl), sodium hydroxide (NaOH), edetate disodium (EDTA), N-hydroxysuccinimide (NHS), tris (hydroxymethyl) aminomethane (Tris), 6-mercapto-1-hexanol (MCH) and Congo red (CR) were purchased from Sinopharm Chemical Reagent Co. Ltd., China. N-(3-Dimethylaminopropyl)-N′ethylcarbodiimide hydrochloride (EDC·HCl) was purchased from Shanghai Medpep Co., Ltd. 4


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Multiwalled carbon nanotubes (MWNTs, diameter 10~20 nm, length 10~30 µm) were purchased from Times Nano, Chengdu, China. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and fullerene (C60) were the products of J&K Scientific Ltd., China. Tween-20 was obtained from Sigma-Aldrich. Projection PET films were obtained from Dongting Screen Factory (Wuxi, China). Nitrogen (N2, purity > 99.9%) was purchased from WISCO Oxygen, Wuhan, China. Silver paste (BQ6880E) was the product of Uninwel, Co., Ltd. Epoxy glue was purchased from Hu'nan Magic Power Industrial Co., China. All chemicals were of analytical grade and used without further purification. All aqueous solutions were prepared using ultrapure deionized water (> 18 MΩ·cm) produced on Heal Force, Nison Instrument Ltd., Shanghai, China. Streptavidin (SA), capture DNA (C-DNA), target DNA (T-DNA), probe DNA (P-DNA) and mismatched sequences (MT1: single-base mismatch, MT3: three-base mismatch) were obtained from Shanghai Sangon Biotechnology Co. (Shanghai, China) and used without further purification. Three DNA sequences related to HBV, HCV and HIV viruses were selected as the analyte models and the sequences are listed below. Target DNA sequences related to HBV, HIV and HCV:34 T-HIV (complementary DNA sequence): 5′-CCA TGA ATT TAG TTG CGC CTG GTC CTT TAA-3′ T-HBV: 5′-ATA CCA CAT CAT CCA TAT AAC TGA AAG CCA-3′ T-HCV (complementary DNA sequence): 5′-ATC TCC AGG CAT TGA GCG GGT TTA TCC ACG A-3′ Thiolated capture DNA sequences: C-HIV: 5′-GCA ACT AAA TTC ATG G-(CH2)3-SH-3′ C-HBV: 5′-TGG ATG ATG TGG TAT-(CH2)3-SH-3′ C-HCV: 5′-CTC AAT GCC TGG AGA T-(CH2)3-SH-3′ Biotinylated probe DNA sequences: P-HIV: 5′-Biotin-TTA AAG GAC CAG GC-3′

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P-HBV: 5′-Biotin-TGG CTT TCA GTT ATA-3′ P-HCV: 5′-Biotin-TCG TGG ATA AAC CCG-3′ Mismatched DNA sequences for T-HBV: MT1-HBV: 5′-ATA CCA CAT CAA CCA TAT AAC TGA AAG CCA-3′ MT3-HBV: 5′-ATA CCA CTT CAT CGA TAT AAC AGA AAG CCA-3′ The aqueous solution for dissolving the capture DNA was a buffer containing 10.0 mM TCEP, 10.0 mM Tris-HCl, 1.0 mM EDTA and 1.0 M NaCl (pH 8.0), and that for the target and probe DNA was 10.0 mM Tris-HCl, 1.0 mM EDTA and 1.0 M NaCl (pH 8.0). The dispersion solution of the streptavidin-labeled MWNTCOOH-CR-C60 PEC bioprobe (SA@MWNTCOOH-CR-C60) was 0.05 wt% Tween-20 in PBS (PBST, 1.0 mM, pH 7.4). The washing buffer for LAPECS was 10.0 mM Tris-HCl (pH 7.4) and the electrolyte solution for 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. All the electrochemical measurements were performed on a CHI 830 analyzer (CH Instruments, Shanghai, China). Scanning electron microscope (SEM) images were collected on a field emission scanning electron microscope (Zeiss, Germany). Transmission electron microscope (TEM) images were characterized by high resolution transmission electron microscopy (HRTEM) (JEOL-2100, Japan). UV-Vis spectra were collected on a UV 2550 spectrophotometer (Shimadzu, Japan). Electrochemical

impedance

spectroscopy

(EIS)

was

carried

out

with

a

Model

283

potentiostat/galvanostat and a Model 5210 lock-in amplifier powered by Powersuit software (EG&G, USA). The electrode system contained a GPETFE working electrode, a platinum wire counter electrode and a potassium chloride (KCl) saturated calomel reference electrode (SCE). All the photocurrent responses were collected at open circuit potentials. A green laser pen with a power of 50 mW at 525 nm and a diameter of ~ 2.5 mm for the illumination area was used as the light source (Figure S2).

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Construction of LAPECS for DNA Sensing. Construction of the LAPECS for the detection of single and multiple DNA sequences using the all-carbon PEC bioprobe and the GPETFE substrate was performed according to the procedures shown in Figure S3 and Figure S4. The corresponding experimental details are also presented in Supporting Information.

Figure 2. (A) UV-Vis spectra of 0.1 mg/mL SA in 0.01 M PBS (pH 7.4) (a) and the supernatant of the SA@MWNTCOOH-CR-C60 solution for the first centrifugation-based washing process (b). (B-D) TEM images of MWNTCOOH (B), the MWNTCOOH-CR-C60 nanohybrid (C) and the SA@MWNTCOOHCR-C60 bioprobe (D).

RESULTS AND DISCUSSION Characterizations of SA@MWNTCOOH-CR-C60. The MWNTCOOH-CR-C60 nanohybrid has been proved to be a promising PEC sensing material with the merits of easy synthesis, facile derivation, good biocompatibility and excellent dispersion stability in aqueous solutions.33 Moreover, the synergetic roles of the three components in the nanohybrid, i.e. C60 is the main photovoltaic material, MWNTCOOH acts as an efficient carrier of C60 and biorecognition molecules and CR is essential to the solution dispersion and high conversion efficiency of C60, endow MWNTCOOH-CR-C60 with high visible light conversion efficiency. 33 This unique property enables the employment of mono-color semiconductor

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laser light sources with high power density and small light beam sizes, which is especially suitable for constructing high throughput PEC sensing systems by the light addressing strategy. Here, through a similar covalent modification process,33 streptavidin can be conveniently fixed on the surface of MWNTCOOH-CR-C60 with the aid of EDC and NHS, producing the SA@MWNTCOOH-CR-C60 bioprobe. As shown in Figure 2A, SA exhibits a strong UV-Vis absorption at 260 nm in 0.01 M PBS (pH 7.4) (curve a), which almost disappears for the supernatant of the centrifuged SA@MWNTCOOHCR-C60 solution during the first washing process after covalent reaction (curve b). This result demonstrates the efficient fixation of SA on MWNTCOOH-CR-C60. TEM images indicate that the MWNTCOOH-CR-C60 nanohybrid has a tubular structure similar to MWNTCOOH, but possesses shorter length and is decorated with some granular C60 nanoparticles along with its sidewall (Figure 2B and 2C). Apparently, the covalent binding of SA hardly influences the morphology of MWNTCOOHCR-C60 (Figure 2D).

Our previous work revealed that the PEC response of the MWNTCOOH-CR-C60 nanohybrid on GPETFE was apparently higher than that on indium-tin oxide (ITO) slides.32 This property, together with the advantages of easy fabrication, good flexibility, large size and facile modification by thiols, demonstrates that GPETFE is an ideal substrate electrode for LAPECS. Moreover, as shown in Figure 3A, the labeling of MWNTCOOH-CR-C60 with SA only causes a slight decline of its photocurrent. The produced SA@MWNTCOOH-CR-C60 bioprobe has good storage stability and 95.9% of its initial signal was retained after the storage at 4 °C under darkness for 4 weeks. Besides, the photo-stability of the SA@MWNTCOOH-CR-C60 bioprobe is also satisfactory (Figure 3B). In the light of the wide applications of affinity reactions in biosensing, the SA@MWNTCOOH-CR-C60 bioprobe may selectively bind with any biotin-labeled species, which endows this probe with an outstanding feature of versatility and avoids the preparation of multiple probes for multiplexed detection.

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Figure 3. (A) Photocurrent responses of GPETFE in 0.1 M PBS (pH 7.4) containing 0.1 M AA when modified by the MWNTCOOH-CR-C60 hybrid (a) and by the SA@MWNTCOOH-CR-C60 bioprobe after a storage period of 0 (b), 1 (c), 2 (d), 3 (e) and 4 weeks (f). (B) Photocurrent responses of the bioprobe with frequent on/off irradiation by the light source.

Construction of LAPECS. Electrochemical impedance spectroscopy (EIS) was utilized to monitor the construction process of the LAPECS. Figure 4A shows the Nyquist plots of different electrodes using [Fe(CN)6]3/4- as the redox probe. The semicircle of the bare GPETFE is small (curve a), indicating a fast electron transfer rate of [Fe(CN)6]3/4- on GPETFE. However, the semicircle is apparently enlarged when C-HIV is modified on the GPETFE through Au-S bonds (curve b), demonstrating the successful assembly of the thiolated capture DNA. The diameter of the semicircle continuously increases after the further modification with MCH (curve c), T-HIV (curve d) and P-HIV (curve e). Notably, the diameter of the semicircle conversely decreases after the binding of the SA@MWNTCOOH-CR-C60 bioprobe

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(curve f), which is a direct evidence for the enhanced electron transfer by the all-carbon bioprobe.33 The photocurrent responses of different electrodes further prove the successful construction of the LAPECS, i.e., the photocurrent continuously decreases in the construction process until the immobilization of the SA@MWNTCOOH-CR-C60 bioprobe (Figure 4B and Figure S5). Therefore, the low background photocurrent of the substrate electrode and the high conversion efficiency of the MWNTCOOH-CR-C60 nanohybrid ensure the high signal-to-noise ratio and sensitivity of the LAPECS.

Figure 4. (A) Nyquist plots of 5.0 mM Fe(CN)63-/4- in 1.0 M KCl. (B) Photocurrents of 0.1 M AA in 0.1 M PBS (pH 7.4) on different electrodes: (a) GPETFE; (b) C-HIV/GPETFE; (c) MCH/C-HIV/GPETFE; (d) T-HIV/MCH/C-HIV/GPETFE; (e) P-HIV/T-HIV/MCH/C-HIV/GPETFE; (f) SA@MWNTCOOHCR-C60/P-HIV/T-HIV/MCH/C-HIV/GPETFE. The concentrations of C-HIV, MCH, T-HIV, P-HIV and the SA@MWNTCOOH-CR-C60 bioprobe for constructing the sensor are 1.0 µM, 5.0 mM, 1.0 nM, 1.0 µM and 1.0 mg/mL (for C60), respectively.

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The successful construction of the LAPECS was also evidenced by SEM images. As indicated in Figure 5, after the modification of C-HIV, T-HIV and P-HIV on a GPETFE by hybridization reactions, the surface of the GPETFE becomes obscure due to the covering by biomolecules. Tubular structures of MWNTCOOH-CR-C60 are clearly observed on the GPETFE when the construction and detection processes of the LAPECS were finished (Figure 5C). Moreover, the higher concentration of T-HIV causes a higher loading of MWNTCOOH-CR-C60 (Figure 5D), and the probe was randomly and homogeneously dispersed on the surface of the substrate electrode (Figure S6 and S7). These results verify the good reliability of coupling bio-recognition systems with affinity reactions for versatile PEC biosensing.

Figure

5.

SEM

images

of

GPETFE

(A),

P-HIV/T-HIV/MCH/C-HIV/GPETFE

(B)

and

SA@MWNTCOOH-CR-C60/P-HIV/T-HIV/MCH/C-HIV/GPETFE (C-D). The concentrations of T-HIV for Parts C and D are 1.0 pM and 0.01 µM, respectively. Scale bars are 100 nm.

Optimization of LAPECS. As the length and melting temperature (Tm) of the three target DNA sequences are close, the hybridization conditions of these DNA should be similar to each other.34 Thus, the construction conditions were optimized by using HBV as a model. In previous works,35,36 the employment of mixed self-assembled monolayers (SAMs) of thiolated capture DNA and MCH has been proved to be a facile approach to control density and minimize nonspecific adsorption of DNA on gold substrate electrodes. Therefore, we employed a similar approach for constructing the LAPECS. The 11



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results showed that the blocking time of MCH had great influences on the background and selectivity of the LAPECS (Figure 6A), i.e., the photocurrents of both the target and the background apparently decrease with the increase of blocking time from 1.0 to 6.0 h and tend to be stable thereafter. A similar trend is observed for the influence of MCH concentration (Figure 6B), i.e., the photocurrents drop rapidly at first and then reach a plateau at a concentration of 5.0 mM MCH. Both results may indicate the formation of saturated MCH self-assembled monolayers on the GPETFE. Therefore, a blocking period of 6.0 h and a concentration of 5.0 mM MCH were selected as the optimal conditions for the multiplexed DNA detection by the LAPECS. As for some other experiment conditions such as hybridization/affinity reaction time and temperature, they were referred to previous literatures.37,16

Figure 6. Influences of blocking time (with 1.0 mM MCH) (A) and concentration of MCH (with 6.0 h block time) (B) on the photocurrent responses of the LAPECS for the detection of 0.0 and 0.01 µM THBV.

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PEC Detection of Single-Component DNA Sequence by LAPECS. In order to evaluate the analytical performance of the LAPECS for DNA sensing, the detection of single-component DNA sequence by the LAPECS was performed. For the PEC sensing of T-HBV, the LAPECS works in a manner that C-HBV was immobilized on all sensing areas of one LAPECS and T-HBV at different concentrations were dropped on different sensing areas of the LAPECS (Figure S3). Through this approach, T-HBV with six concentrations for calibration can be detected by two sensors. As indicated in Figure 7A, the photocurrent increases gradually with the concentration of T-HBV, and the photocurrent difference between the target and the background (∆I) shows a good linear relationship with the logarithm of THBV concentration (logCT-HBV) in the range from 1.0 pM to 0.1 µM (Figure 7B). The detection limit is 0.7 pM, which is better than some fluorescence methods for multiplexed DNA detection.38,39 As mentioned above, the GPETFE was separated into four equal areas (4 mm×4 mm) by insulative paint line barriers, and the light leakage for false positive signal can be completely avoided by placing the small light beam (~2.5 mm in diameter) on the central area of each sensing zone (Figure S2).

Figure 7. Photocurrent responses (A), calibration plot (B) and selectivity evaluation (C) for the detection of T-HBV by the LAPECS (n = 3). The concentrations of T-HBV on Part A are 0.0, 0.0007, 0.001, 0.01, 0.1, 1.0, 10.0, 100.0 nM (from top to bottom), and the concentrations on Part C are 0.01 µM for all the tested DNA sequences. Insets of Part B show the schematic patterns for the detection of THBV with varied concentrations on different sensing areas of two LAPECS for calibration.

To evaluate the selectivity of the LAPECS, the photocurrent responses of different DNA sequences

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on the above LAPECS were investigated (Figure 7C). The results indicate that the sensor exhibits a much higher photocurrent for T-HBV compared with T-HIV and T-HCV at the same concentration of 0.01 µM, and the responses of T-HIV and T-HCV are very close to the background. Moreover, the response of T-HBV is obviously distinguished from that of the mismatched sequences (MT1 and MT3). We also examined the analytical performance of the LAPECS for the detection of T-HIV and T-HCV, and very similar results were observed in terms of sensitivity and selectivity (Figure S8 and S9). Further experiments demonstrated that the presence of 0.01 µM T-HIV had little influence on the detection of THCV in the range from 1.0 pM to 0.1 µM, reflected by the small deviation between the calibration plots in the absence and the presence of the interferent (Figure S10). These results clearly indicate the good sensitivity and selectivity of the LAPECS for the detection of individual DNA sequences.

PEC Detection of Multiple DNA Sequences by LAPECS. Besides the detection of individual DNA sequences with varied concentrations, the LAPECS can also work in a manner that enables multiplexed DNA detection using one sensor (Figure S4). In this case, the capture DNA sequences of different targets were immobilized on different sensing areas of one LAPECS, and a mixture solution of different DNA targets at the same concentration, i.e., T-HIV, T-HBV and T-HCV, was incubated with each sensing area of the sensor. Then, different target DNA sequences can be selectively captured and detected on different sensing areas by the light addressing strategy. The results indicate that the LAPECS has a very similar analytical performance for the multiplexed detection of T-HIV, T-HBV and T-HCV, possessing a similar calibration range of 1.0 pM ~ 0.01 µM and a low detection limit of 0.7 pM (Figure 8A and 8B). It should be pointed out that although only three DNA sequences are employed as the target analytes here, the detection throughput may be easily expanded to tens to hundreds of samples by simply enlarging the size of the substrate electrodes or reducing the size of the sensing arrays and the light beam. Moreover, the coupling of bio-recognition systems with affinity reactions for the LAPECS allows the high throughput detection of multiple DNA targets in one sample with varied concentrations,

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which may not only simplify the detection process but also provide comprehensive fingerprint-like information of multiple biomarkers for the diagnosis of a certain disease by just one test (Figure 8C-E). Different from the LAPS, the LAPECS can work in a manner similar to solar cells (Figure S11), and thus allow the development of portable, self-driving and high throughput PEC biosensing systems by employing simple and integrated current test instruments like galvanometers, multimeter, glucose meters or even paper supercapacitors. 21

Figure 8. Photocurrent responses (A) and calibration plots (B) for the multiplexed detection of T-HIV (a), T-HCV (b) and T-HBV (c) (at the same concentration) by the LAPECS. Parts C-E show the photocurrent responses of a mixture of multiple DNA targets with varied concentrations. The concentrations for the three target DNA on Part A are 0.0, 0.001, 0.01, 0.1, 1.0, 10.0 nM (from top to bottom). Inset of Part B shows the sensing array pattern for the multiplexed detection of three DNA sequences on different areas of a LAPECS.

Stability and Reproducibility. The stability of the LAPECS was examined using T-HBV as a model, i.e., a batch of MCH/C-HBV/GPETFE were stored in a buffer containing 10.0 mM Tris-HCl, 1.0 mM EDTA and 1.0 M NaCl (pH 8.0) at 4 °C under darkness for different periods prior to the PEC detection

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of 0.01 µM T-HBV. The photocurrent response retained 95.9% and 91.9% of its initial response after the storage of 2 and 3 weeks, respectively (Figure S12A). Here, the stability test was performed by using the same solution of the SA@MWNTCOOH-CR-C60 probe for all the samples. These results, together with the data of Figure 3A, indicate the good stability of both MCH/C-HBV/GPETFE and the all-carbon PEC bioprobe. The LAPECS also possessed quite good reproducibility, and a low relative standard deviation (RSD) of 2.9% was obtained for the parallel measurements of 0.01 µM T-HBV by six different sensors (Figure S12B).

Figure 9. Multiplexed detection of 1.0 nM T-HCV, 1.0 nM T-HBV and 1.0 nM T-HIV in serum samples by the LAPECS.

Detection of DNA by LAPECS in Simulated Serum Samples. To evaluate the applicability of the sensor for real samples, the LAPECS was employed for the multiplexed detection of T-HCV, T-HBV and T-HIV in 100-fold diluted serum samples by using a standard addition method34,40 (Figure 9). Compared with the responses in the buffer solution, the photocurrents of all the three DNA targets in the serum samples are enlarged but the increments of the photocurrents are small (

Ultrasensitive Photoelectrochemical Biosensing of Multiple

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