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. 2022 Oct 14;7(42):37499–37508. doi: 10.1021/acsomega.2c04280

Trace Analysis of Emerging Virus: An Ultrasensitive ECL-Scan Imaging System for Viral Infectious Disease

Yunxia Wu ‡, Ronghua Yang §, Qikang Wu ‡, Mingxing Huang ∥, Bowen Shu †, Wenjie Wu †, Baoqing Sun ⊥,*, Jinyu Xia ∥,*, Xiaodong Chen ‡,*, Yuhui Liao †,∥,*
PMCID: PMC9609065  PMID: 36312431

Abstract

graphic file with name ao2c04280_0008.jpg

Emerging infectious diseases have brought a huge impact on human society in recent years. The outbreak of Zika virus (ZIKV) in the Americas resulted in a large number of babies born with microcephaly. More seriously, the Coronavirus Disease 2019 (COVID-19) was globally spread and caused immeasurable damages. Thus, the monitoring of highly pathogenic viruses is important to prevent and control emerging infectious diseases. Herein, a dendritic polymer probe-amplified ECL-scan imaging system was constructed to realize trace analysis of viral emerging infectious diseases. A dendritic polymer probe was employed as the efficient signal emitter component that could generate an amplified ECL signal on the integrated chip, and the signal was detected by a single-photon level charge coupled device-based ECL-scan imaging system. With this strategy, the ZIKV in a complex system of blood, urine, and saliva was detected. The results indicated that a high sensitivity of 50 copies and superior specificity were achieved. Furthermore, this strategy realized highly sensitive detection (10 copies) of the S and N protein gene sequence of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-Cov2) and spiked pseudovirus samples. Thus, the dendritic polymer probe-amplified ECL-scan imaging system suitably met the strict clinical requirements for trace analysis of an emerging virus, and thus has the potential to serve as a paradigm for monitoring emerging infectious diseases.

Introduction

With the development of economic globalization, the population density and mobility have greatly increased in recent years. The accompanying problem is the safety, prevention, and control of highly pathogenic microorganisms. In 2015, the Zika virus (ZIKV),1−3 an Aedes mosquito-borne flavivirus,4,5 was widespread in the Americas6−8 and was declared as a Public Health Emergency of International Concern by World Health Organization (WHO).9−11 ZIKV spreads in a variety of ways, including by infected mosquito bite.12,13 It also can be transmitted by mother-to-fetus transmission,14 sexual contact,15,16 or blood transfusion.17 Meanwhile, it has been proven that ZIKV infection was a main cause of Guillain-Barre syndrome,18,19 congenital microcephaly,20 and neurological defects in newborns.21 Also ZIKV caused huge economic losses and health threats in the Americas. Fortunately, the ZIKV did not induce global spread.

More seriously, the Coronavirus Disease 2019 (COVID-19)22,23 was spread globally and caused immeasurable damages. The infection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-Cov2)24,25 could cause the clinical symptoms of fever, chest pain, chills, rapid heartbeat, breathing difficulties, pneumonia, and kidney failure.26−33 More than 250 million patients were confirmed to be infected with SARS-Cov2, which could spread though droplet transmission induced by coughing, sneezing, and touch, and the virus caused 5.1 million deaths (according to the statistics of Johns Hopkins university until Nov-16-2021). The mortality due to this virus is more than 10% in some European countries such as France,34,35 England,36 and Italy,37,38 and the SARS-Cov2 has brought unprecedented influence on human society. However, biomedical analysis and accurate diagnosis of a viral emerging infectious disease are still unreliable because infected patients can be asymptomatic or present symptoms like other febrile illnesses. Thus, the monitoring of highly pathogenic virus is of great significance to the prevention and control of emerging infectious disease.

Electrochemiluminescence (ECL) is a versatile and powerful analytical technique, and since the first ECL reactions were reported in the 1960s,39,40 ECL has been widely applied in various fields, including biological clinical and applications.41 ECL is mainly a luminescence process caused by the relaxation of electronically excited products to the ground state after an electrochemical reaction, including an electrochemical excitation step and a photoemission process. Specifically, the electrochemical reaction provides the active intermediate and the excited state photophore; in the subsequent luminescence relaxation process, the excited photophore returns to the ground state and emits an ECL signal.42 Compared to other optical-based analysis methods, ECL has the following advantages. First, no external light source is required, and background noise from autofluorescence or scattered light is completely avoided. Second, the potential applied to the electrode allows for controlled ECL signal release. Third, the method has a fast response, high sensitivity, wide detection range, and low cost, and only small instruments are required.43−46

Herein, a dendritic polymer probe-amplified ECL-scan imaging system was constructed to realize trace analysis of viral emerging infectious disease (Figure 1). The dendritic polymer probe was employed as the efficient signal emitter component that could generate an amplified ECL signal on the integrated chip, and the signal was detected by a single-photon level charge coupled device-based ECL-scan imaging system. With this strategy, the ZIKV in the complex system of blood, urine, and saliva was detected. The results indicated that a high sensitivity of 10 copies and superior specificity were achieved. Furthermore, this strategy simultaneously realized ultrasensitive detection of the S and N protein gene of the SARS-Cov2 and spiked pseudovirus samples. Therefore, the dendritic polymer probe-amplified ECL-scan imaging system suitably met the strict clinical requirements for trace analysis of a highly pathogenic virus, and thus has the potential to be a new paradigm for emerging infectious diseases.

Figure 1.

Figure 1

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Design of dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system. (A) Formation process of dendritic polymer probe. (B) ECL detection process of dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system. (C) Structure of ECL-scan imaging system. (D) Structure of ECL-scan imaging system. (E) Details of ECL chip.

Results and Discussion

Design of Dendritic Ru(bpy)32+-Polymer Amplified ECL-Scan Imaging System

The dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system was constructed for trace analysis of viral emerging infectious diseases. In this strategy, the dendritic polymer probe was used as a component that could generate an amplified ECL signal on an integrated chip. The formation process of the dendritic polymer probe is shown in Figure 1A. The dendritic polymer was connected to a DNA recognition domain by the addition reaction of a sulfydryl double bond, and the detailed process is shown in Figure S1. Then, the ECL luminophore of Ru(bpy)32+ was linked to the dendritic polymer that could generate an intense ECL signal. The virus sample was dissociated to release RNA which was immobilized by capture probe (Figure 1B), and the dendritic polymer probe was subsequently added to form the complete ECL generation complex with streptavidin-labeled magnetic beads. The ECL signal was definitively detected by a single-photon level charge coupled device-based ECL-scan imaging system. The structure of the ECL-scan imaging system and the ECL chip are shown in Figure 1C,D. With this strategy, the ZIKV in the complex system of blood, urine, and saliva was detected. Furthermore, this strategy simultaneously realized highly sensitive detection of the S protein gene sequence of SARS-Cov2 and spiked samples.

Characterization of Dendritic Ru(bpy)32+-Polymer

The molecular structure and the activation process of Ru(bpy)32+ are shown in Figure 2A. Ru(bpy)32+ was synthesized following the protocol in our previously published works,47,4849 and the synthetic route is listed in Figure S2. The fluorescence and absorption characterization in Figure 2B,C indicated that the synthesis of Ru(bpy)32+ and the activation were feasible, and the results were consistent with previous results. Then, we verified the connection between the dendritic Ru(bpy)32+-polymer and the DNA recognition domain. The results in Figure 2D,E confirmed the feasibility of the connection between the dendritic Ru(bpy)32+-polymer and the DNA recognition domain. The AFM results in Figure 2F showed that the morphology of the dendritic Ru(bpy)32+-polymer probe appeared to be an irregular sheet distribution. The above characterization results proved the feasibility of dendritic Ru(bpy)32+-polymer probe. Meanwhile, the stability of the dendritic Ru(bpy)32+-polymer probe was investigated. The results in Figures S3, S4, S5, and S6 indicated that the dendritic Ru(bpy)32+-polymer probe could remain stable at the high temperature of 95 °C, at the pH in the range of 5.0–8.5, in different buffers (PBS, SSC, and Tris-HCl), and in the complex biosystem of blood, urine, and saliva.

Figure 2.

Figure 2

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Characterization of dendritic Ru(bpy)32+-polymer. (A) Molecular structure and activation process of Ru(bpy)32+. (B) Fluorescence characterization of Ru(bpy)32+ and the activated Ru(bpy)32+. (C) Absorption characterization of Ru(bpy)32+ and the activated Ru(bpy)32+. (D) Absorption characterization of dendritic Ru(bpy)32+-polymer probe. (E) Excitation and emission of dendritic Ru(bpy)32+-polymer probe. (F) Morphology of dendritic Ru(bpy)32+-polymer probe under atomic force microscope (AFM).

Principle Validation, Optimization, and Sensitivity of ECL-Scan Imaging System

The ECL-scan imaging system comprised the single-photon level charge coupled device, the microscope system, and the ECL system. The ECL signal that was generated by the dendritic Ru(bpy)32+-polymer probe was actuated by the ECL system, which was comprised the ECL chip and the high-precision motorized positioning systems (X and Y axis direction). The ECL signal was first focused by the objective lens and projection lens of the microscope, and then collected by the single-photon level charge coupled device. Under the light field, the complete ECL detection complex with streptavidin-labeled magnetic beads emerged as a spherical morphology (Figure 3A). While the electric field was added, the ECL signal was generated on the ECL chip and detected by the single-photon level charge coupled device under the dark field. The results in Figure 3B showed that intense ECL signal was generated on the surface of streptavidin-labeled magnetic beads. The particle size and zeta potential of streptavidin-labeled magnetic beads showed obvious distinction after the target was present (Figure 3C). The time (exposure time of the ECL-scan imaging system) of data acquisition was optimized. The results in Figure 3D indicated that the ECL intensity increased with the extension of time, and reached a plateau at the time point of 12 s, which means at this time point, the sensitivity of the ECL system is the highest, and the detection time is the shortest. Therefore, we select 12 s as the optimal time of data acquisition. Furthermore, the key factors of experiment were evaluated in Figure 4A,B,C. The results indicated that the optimal experiment conditions of hybridization time, electrode voltage, and washing time were set as 15 min, 2.4 V, and 4 times.

Figure 3.

Figure 3

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Principle validation of dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system. (A) ECL generation complex with streptavidin-labeled magnetic beads under light field. Scale bar: 10 μm. (B) ECL signal detection of ECL generation complex under dark field. Scale bar: 10 μm. (C) Particle size and zeta potential of streptavidin-labeled magnetic beads and ECL generation complex with streptavidin-labeled magnetic beads. (D) Optimization of the time of data acquisition.

Figure 4.

Figure 4

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Optimization, sensitivity, and specificity of dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system. (A). Optimization of hybridization time. (B) Optimization of electrode voltage. (C) Optimization of washing time. (D) Sensitivity and linear analysis of dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system. (E) Specificity of dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system with S1 and S2. (F) Specificity of dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system with random sequence of RS1 and RS2.

With the optimal experiment conditions, we evaluated the sensitivity of dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system with the sequence from the HBsAg gene of the Hepatitis B virus (HBV). The sequence was listed in Table S1. The results in Figure 4D revealed that the dendric Ru(bpy)32+-polymer probe reached a high sensitivity of 1 fmol, and provided a wide linear response range from 1 fmol to 104 fmol. Therefore, the excellent luminescence of the dendric Ru(bpy)32+-polymer probe provided the foundation for clinical detection and diagnosis. Furthermore, we investigated the specificity of dendric Ru(bpy)32+-polymer probe by comparing the ECL intensity between the specific sequence and random sequences. The sequence was listed in Table S1. The results in Figure 4E,F indicated that only the specific HBV sequence resulted in intense ECL; random sequences showed essentially the same ECL intensity as that of the control group. This result verifies the specificity of the dendric Ru(bpy)32+-polymer probe.

Application of Dendritic Ru(bpy)32+-Polymer Amplified ECL-Scan Imaging System for Zika Virus

Zika virus (ZIKV), an Aedes mosquito-borne flavivirus, was widespread in the Americas in 2015. It was declared as a Public Health Emergency of International Concern by World Health Organization (WHO) and caused huge economic losses and health threats in the Americas. Herein, we expanded the application of dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system for detection of ZIKV. The conserved sequence from the ZIKV NS1 gene was designed, and the genomes of the ZIKV strains and the other flaviviruses used in the present study were downloaded from the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/genome/viruses/variation/Zika/), and aligned with the analysis software of MEGA 7. Conserved ZIKV-specific sequences that were divergent from other flaviviruses were identified. The sequence of ZIKV and other flavivirus strains, sequence of capture probe and signal probe (Table S2), kinetic parameters, and hybrid structure are shown in Figure 5.

Figure 5.

Figure 5

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Sequence of ZIKV and other flavivirus strains, sequence of capture probe and signal probe, kinetic parameters, and hybrid structure.

With the designed sequence, we assessed the sensitivity and specificity of the dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system for ZIKV. First, we verified the specificity of the dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system with other flaviviruses. The results in Figure 6A indicated that the dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system could specifically respond to ZIKV, and the other flavivirus group did not give out the ECL signal that could be differentiated from the control group. Therefore, the specificity of dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system obtained a better proof. The sensitivity was also evaluated in Figure 6A, and the results of low concentration were shown in Figure S7. The results revealed that dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system reached a satisfactory sensitivity of 50 copies for the ZIKV samples, which is higher for conventional methods including reverse transcription (RT)-PCR (100 copies), nucleic acid sequence-based amplification (NASBA) (100 copies), and ELISA (10–30 ng/mL).

Figure 6.

Figure 6

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Performance Evaluation of Dendritic Ru(bpy)32+-Polymer Amplified ECL-scan Imaging System for ZIKV. (A) Sensitivity and specificity of dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system for ZIKV. (B) Detection of blood samples of mice. (C) Detection of urine samples of mice. (D to F) Detection of ZIKV spiked human blood samples. (G to I) Detection of ZIKV spiked human urine samples.

Subsequently, the blood and urine samples from the ZIKV infected mouse mode were collected and detected. The blood samples and urine samples were treated by SDS buffer by five times’ the volume. The blood sample from the mouse that infected the ZIKV was first detected. The results in Figure 6B revealed that the dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system achieved a stable signal of the detection process. The urine sample from the infected mouse was collected by a simple device reported by our group. The results in Figure 6C indicated that the dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system could stably respond to all the ZIKV samples from the urine of infected mouse. Furthermore, the spiked samples in human blood and urine was detected to verify the feasibility for ZIKV detection in human samples. The results in Figure 6D to F (spiked blood samples) and Figure 6G to I (spiked urine samples) proved the capacity of dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system for the detection of human samples. Thus, it can be proven that this dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system possesses the ability to detect ZIKV from complex environments.

Application of Dendritic Ru(bpy)32+-Polymer Amplified ECL-Scan Imaging System for SARS-Cov2

The COVID-19 spread globally and caused immeasurable damages. More than six million patients were confirmed to be infected with SARS-Cov2 until Jun-4-2020. The high mortality brought unprecedented influence on human society. Thus, the monitoring of a highly pathogenic virus is of great significance to the prevention and control of emerging infectious diseases. In this section, the sequence of S protein and N protein gene were chosen as the instances for detection of SARS-Cov2 with the dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system. The selected sequence of S protein and N protein gene, the capture and signal probe (Table S3), kinetic parameters, and hybrid structure were listed in Figure 7A. With the synthetic sequence, we examined the sensitivity and specificity of the dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system for SARS-Cov2. The results in Figure 7B,C indicated that dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system achieved a consistent performance with ZIKV detection. The sensitivity of 10 copies and preferable specificity were achieved with the spiked pseudovirus samples, and the results of low concentration were shown in Figures S8 and S9. The detection of spiked saliva and blood samples also confirmed the outstanding capacity of dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system for detection in human samples.

Figure 7.

Figure 7

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Performance evaluation of dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system for SARS-Cov2. (A) Selected sequence of S protein and N protein gene, the capture and signal probe, kinetic parameters, and hybrid structure. (B) Sensitivity of dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system for SARS-Cov2. (C) Specificity of dendritic Ru(bpy)32+-polymer amplified ECL-scan imaging system for SARS-Cov2. (D) Detection of spiked saliva samples. (E) Detection of spiked blood samples.

Conclusions

A dendritic polymer probe-amplified ECL-scan imaging system was constructed to realize trace analysis of a viral emerging infectious disease. A dendritic polymer probe was employed as the efficient signal emitter component that could generate an amplified ECL signal on the integrated chip, and the signal was detected by a single-photon level charge coupled device-based ECL-scan imaging system. With this strategy, the ZIKV in the complex system of blood, urine, and saliva was detected. The results indicated that a high sensitivity of 50 copies and superior specificity were achieved. Furthermore, this strategy simultaneously realized highly sensitive detection (10 copies) of the S protein gene sequence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-Cov2) and spiked samples. Therefore, the dendritic polymer probe-amplified ECL-scan imaging system suitably met the strict clinical requirements for trace analysis of highly pathogenic viruses, and thus has the potential to serve as a liquid biopsy paradigm for monitoring viral emerging infectious diseases.

Experimental Section

Reagents

All the chemical reagents for the synthesis and activation of Ru(bpy)32+, such as cis-bis (2,2′-bipyridine) dichlororuthenium(II), 2,2′-bipyridine-4,4′-dicarboxylic acid, N,N′-dicyclohexylcarbodiimide (DCC), sodium hexafluorophosphate, N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and sodium borate were obtained from Alfa Aesar Co., Ltd. The single-photon level charge coupled device was customized by Tucsen Photonics Co., Ltd. Streptavidin magnetic beads were synthesized by New England BioLabs. Reagents related to electrophoresis and DNA probes were purchased from Shanghai Sangon Biotechnology Co. Ltd. SYBR I and SYBR II were purchased from APExBIO (Houston, USA). Reagent grade dendritic polymer was purchased from Sigma-Aldrich and was used without further purification except where noted. Invitrogen synthesized all oligonucleotides. Glass botttom dishes and centrifuge tubes were obtained from NEST Biotechnology (Wuxi, China).

Synthetic Routes of Ru(bpy)32+

The synthesis routes of active Ru(bpy)32+ followed the procedure of Ru(bpy)2(dcbpy)(PF6)2 and the activation step of the carboxyl on Ru(bpy)32+. The protocols were based on the previous works.47,48 The detailed synthetic routes of Ru(bpy)32+ are shown in Figure S2. The reactant of cis-dichlorobis(2,2′-bipyridine)ruthenium(II) with an excess of the corresponding 2,2′-bipyridine-4,4′-dicarboxylic acid (1:1.2) was refluxed in an ethanol/water (80%) mixture, and the mixture was incubated at 80 °C for one night to synthesize Ru(bpy)2(dcbpy)(PF6)2. Then, the solution was subsequently cooled and acidified (pH = 4.4) to crystallize out the unreacted 2,2′-bipyridine-4,4′-dicarboxylic acid, which was eliminated by a qualitative filter paper based suction filter. The precooled solution of sodium hexafluorophosphate (NaPF6) was added to the resultant filtrate, isolating the ruthenium complexes as PF6 salts. The mixture was cooled with an ice–water mixture. Then, the crystal products were treated with the freeze-drying process to eliminate the influence of volatile solvent.

Synthetic Routes of Dendric Ru(bpy)32+-Polymer

The core of dendric polymer was obtained from Sigma, then modified with amino via the lipid reaction. The mixture of maleimide-labeled DNA and the amino-labeled dendric polymer (the molecular ratio between maleimide-labeled DNA and the amino-labeled dendric polymer was set as 20:1) was incubated for 12 h at 37 °C with stirring. Then, the dissociative DNA was eliminated by ultrafiltration to increase the sensitivity and signal-to-noise ratio. The purified products were mixed with carboxyl-activated Ru(bpy)32+ at the molecular ratio of 1:5000, and incubated at 37 °C for 12 h with stirring. The products were purified by ultrafiltration to eliminate free Ru(bpy)32+.

Dissociation Process of Virus

The virus samples were cracked by a commercial virus-lysis kit, the key constituent was sodium dodecyl sulfate (SDS), and the solution was heated to 60 °C for 5 min. Then, 10 μL of magnetic beads (1 mg/mL) labeled with the capture probe was added in each sample for 10 min. The mixture was processed by magnetic separation. The captured products were collected for later ECL steps.

ECL Process

The signal probe, composed of the dendric Ru(bpy)32+-polymer and DNA recognition domain, was employed as the ECL-generating group. The signal probe and NHS-activated dendric Ru(bpy)32+-polymer were incubated at room temperature (R.T.) in a 1:1 molar ratio for covalent linkage. Meanwhile, the biotin labeled capture probes (10 μM) were bound to the magnetic beads (104/mL) labeled with the streptavidin through R.T. co-incubation. Then the sample containing target nucleic acid was added into the excess mixture of signal probe-dendric Ru(bpy)32+-polymer and capture probe-magnetic beads (the molar ratio of the signal probe and the capture probe was 1:1) for specific binding, and the classical “sandwich” structure of ECL was constituted. A gradient cooling process would be more effective for formation. After the cleaning steps by magnetic bead adsorption and PBS washing for 4 times, the ECL signal of the “sandwich” structure was detected in the presence of the coreactant of tripropylamine (TPA, 10 μM). Typically, the electrode voltage and the accepted wavelength range of the ECL instrument was set as 2.4 V, and 300–800 nm, respectively. The parameters such as exposure time, effective area, pixel size, and resolution of the CCD system was set as 12 s, 11.1 × 6.3 mm2, 2.9 μm (H) × 2.9 μm (V), and 3840 (H) × 2160 (V), respectively.

Acknowledgments

This work was supported by the National Key R&D Program of China (2021YFC2302200), Natural Science Fund of Guangdong Province for Distinguished Young Scholars (22050000282), National Natural Science Foundation of China (81972019, 21904145, 82102360, 81772136), Special Fund of Foshan Summit Plan (2020B019, 2020B012, 2020A015), Zhongnanshan Medical Foundation of Guangdong Province (ZNSA-2021012), Training project of National Science Foundation for Outstanding/Distinguished Young Scholars of Southern Medical University (C620PF0217), China Postdoctoral Science Foundation (2021M690634), Guangdong Basic and Applied Basic Research Foundation (2020A1515110529), Foundation of Foshan City (FS0AA-KJ218-1301-0034, 2018AB003411), Guangzhou Institute of Respiratory Health Open Project (Funds provided by China Evergrande Group, Project No. 2020GIRHHMS04).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c04280.

  • Principle of probe assembly; synthetic routes of Ru(bpy)32+; stability of dendritic Ru(bpy)32+-polymer probe; result details of low concentration samples; sequence of target RNA and probes (PDF)

Author Contributions

# Y.W., R.Y., Q.W., and M.H. contributed equally.

The authors declare no competing financial interest.

Supplementary Material

ao2c04280_si_001.pdf (409KB, pdf)

References

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