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

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 been greatly increased in recent years. The accompanying problem is the safety, prevention and control of the highly pathogenic microorganisms. In 2015, Zika virus (ZIKV), [1][2][3] an Aedes mosquitoborne flavivirus, [4,5] was widespread in the Americas. [6][7][8] And it has been declared as a Public Health Emergency of International Concern by World Health Organization (WHO). [9][10][11] ZIKV spreads in a variety of ways, including infected mosquito bite, [12,13] also can be transmitted from mother-to-fetus transmission, [14] sexual contact, [15,16] or blood transfusion. [17] Meanwhile, it has been proved that ZIKV infection was the main cause of Guillain-Barré syndrome, [18,19] congenital microcephaly, [20] and neurological defects in newborns. [21] And the ZIKV caused the huge economic losses and health threats in the Americas. Fortunately, the ZIKV did not induced the global spreads.
More seriously, the Coronavirus Disease 2019 (COVID-19) [22,23] caused the global spreads and immeasurable damages. The infection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-Cov2) [24][25][26] could cause the clinical symptoms of fever, chest pain, chills, rapid heartbeat, breathing difficulties, pneumonia, and kidney failure. [27][28][29][30][31][32][33][34] More than twenty million patients was confirmed to be infected with SARS-Cov2 (according to the statistics of Johns Hopkins university until Mar-19-2021), which could spreads though droplet transmission induced by coughing, sneezing and touch. The mortality is more than 10 percent in some European countries such as France, [35,36] England [37] and Italy. [38,39] And the SARS-Cov2 has brought unprecedented influence on human society. However, the biomedical analysis and accurate diagnosis of viral emerging infectious disease still facing that the infected patients were asymptomatic or present symptoms like other febrile illnesses. Thus, the monitoring of highly 4 pathogenic virus is of great significance to the prevention and control of emerging infectious disease.
Herein, a dendritic polymer probe-amplified electrochemiluminescence (ECL) [40][41][42] scan imaging system was constructed to realize trace analysis of viral emerging infectious disease ( Figure 1). Dendritic polymer probe was employed as the efficient signal giving-out component that could generate 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 were detected. The results indicated 5 that a high sensitivity of 10 copies and superior specificity were achieved. Furthermore, this strategy simultaneously realized ultrasensitive detection of S and N protein gene of 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 highly pathogenic virus, and thus has the potential to be a new paradigm for the emerging infectious disease.

Reagents.
All the chemical reagents for the synthesis and activation of Ru(bpy)3 2+ , such as cis-Bis ( Reagentgrade dendritic polymer was purchased from Sigma-Aldrich and was used without further purification except where noted. Invitrogen synthesized all oligonucleotides.

Synthetic Routes of Dendric Ru(bpy)3 2+ -polymer.
The core of dendric polymer were 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 hours at 37 ℃ with stir. 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)3 2+ at the molecular ratio of 1:5000, and incubated at 37℃ for 12 hours with stir. The products were purified by ultrafiltration to eliminate free Ru(bpy)3 2+ .

Dissociation Process of Virus and ECL process.
The virus samples were cracked by commercial virus-lysis kit, the key constituent was sodium dodecyl sulfate (SDS), the solution can be heated to 60 ℃ for 5min. Then, the 10 uL magnetic beads (1mg/mL) labeled with capture probe were added in each sample for 10min. The mixture was processed by magnetic separation. The captured products was collected for later ECL steps.
The signal probe, composed of the dendric Ru(bpy)3 2+ -polymer and DNA recognition domain, was employed as the ECL-generating group. The capture probes were bound by the magnetic beads labeled with the streptavidin that could recognize and bind biotin from the capture probe. While 7 the target exists, the classical 'sandwich' structure of ECL was constituted by the capture probe and signal probe. A gradient cooling process would be more effective for formation. After the cleaning steps, the ECL signal was detected in the presence of the co-reactant of tripropylamine (TPA).

Design of Dendritic Ru(bpy) 3 2+ -Polymer Amplified ECL-scan Imaging System.
The dendritic Ru(bpy)3 2+ -polymer amplified ECL-scan imaging system was constructed for trace analysis of viral emerging infectious disease. In this strategy, the dendritic polymer probe was employed as the efficient signal giving-out component that could generate amplified ECL signal on the integrated chip. The formation process of dendritic polymer probe is shown in Figure   1A. The dendritic polymer was connected to DNA recognition domain by addition reaction of sulfydryl-double bond, and the detailed process listed in Figure S1. Then, the ECL luminophore of Ru(bpy)3 2+ was linked to the dendritic polymer that could generate intense ECL signal. The virus samples was dissociated to release RNA which 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 ECLscan imaging system and the ECL chip was shown in Figure 1 C and D. With this strategy, the ZIKV in the complex system of blood, urine and saliva were detected. Furthermore, this strategy simultaneously realized highly sensitive detection of S protein gene sequence of SARS-Cov2 and spiked samples.

Characterization of Dendritic Ru(bpy) 3 2+ -Polymer.
8 The molecular structure and the activation process of Ru(bpy)3 2+ are shown in Figure 2A. The synthesis of Ru(bpy)3 2+ was following the protocol in our previously published works, [43][44][45] and the synthetic route was listed in Figure S2. The fluorescence and absorption characterization in Figure 2B and C indicated that the synthesis of Ru(bpy)3 2+ and the activation was feasible, and the results was consistent with previous results. Then, we verified the connection between the dendritic Ru(bpy)3 2+ -polymer and the DNA recognition domain. The results in Figure 2D and E approved the feasibility of the connection between the dendritic Ru(bpy)3 2+ -polymer and the DNA recognition domain. The AFM results in Figure 2F showed that the morphology of dendritic Ru(bpy)3 2+ -polymer probe appeared irregular sheet distribution. The above characterization results proved the feasibility of dendritic Ru(bpy)3 2+ -polymer probe. Meanwhile, the stability of dendritic Ru(bpy)3 2+ -polymer probe was investigated. The results in Figure S3, S4, S5 and S6 indicated The ECL-scan imaging system was constituted by the single-photon level charge coupled device, the microscope system and the ECL system. The ECL signal that generated by the dendritic Ru(bpy)3 2+ -polymer probe was actuated by the ECL system, which was constituted by the ECL chip and the high-precision motorized positioning systems (X and Y axis direction). The ECL signal was firstly 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 emerge as 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. And the particle size and Zeta potential of streptavidin-labeled magnetic beads showed obvious distinction after the target was present (Figure 3C). The time 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 second. Furthermore, the key factors of experiment was evaluated in Figure 4A, B and C. The results indicated that the optimal experiment conditions of hybridization time, electrode voltage and washing time were set as 15min,

2.3V and 4 times.
With the optimal experiment conditions, we evaluated the sensitivity of dendritic Ru(bpy)3 2+polymer amplified ECL-scan imaging system with the sequence from the HBsAg gene of the amplified ECL-scan imaging system with S1 and S2. F. Specificity of dendritic Ru(bpy)3 2+ -polymer amplified ECL-scan imaging system with random sequence of RS1 and RS2. Table S1. The results in Figure 4D revealed that the dendric Ru(bpy)3 2+ -polymer probe reached a high sensitivity of 1fmol, and provided a wide linear response range from 1 fmol to 10 4 fmol. Therefore, the excellent luminescence of the dendric Ru(bpy)3 2+ -polymer probe provided the foundation for clinical detection and diagnosis.

Hepatitis B virus (HBV). The sequence was listed in
Furthermore, we investigated the specificity of dendric Ru(bpy)3 2+ -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 and 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 dendric Ru(bpy)3 2+ -polymer probe.

Application of Dendritic Ru(bpy) 3 2+ -Polymer Amplified ECL-scan Imaging System for Zika
Virus.  (Table S2), kinetic parameters and hybrid structure are shown in With the designed sequence, we assessed the sensitivity and specificity of dendritic Ru(bpy)3 2+polymer amplified ECL-scan imaging system for ZIKV. Firstly, we verified the specificity of dendritic Ru(bpy)3 2+ -polymer amplified ECL-scan imaging system with other flavivirus. The results in Figure 6A indicated that dendritic Ru(bpy)3 2+ -polymer amplified ECL-scan imaging system could specifically response to ZIKV, and the other flavivirus group did not give out the ECL signal that could differentiate from control group. Therefore, the specificity of dendritic Ru(bpy)3 2+ -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.

13
The results revealed that dendritic Ru(bpy)3 2+ -polymer amplified ECL-scan imaging system reached a satisfactory sensitivity of 50 copies for ZIKV 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' volume. The blood sample from the mouse that infected ZIKV was firstly detected. The results in Figure 6B revealed that the dendritic Ru(bpy)3 2+ -polymer amplified ECL-scan imaging system achieved a stable signal of the detection process . The urine sample from infected mouse was collected by a simple device reported by our group. The results in Figure 6C indicated that the 14 dendritic Ru(bpy)3 2+ -polymer amplified ECL-scan imaging system could stably response 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)3 2+ -polymer amplified ECL-scan imaging system for detection human samples. Thus, it can be proved that this dendritic Ru(bpy)3 2+ -polymer amplified ECL-scan imaging system possesses the ability to detect ZIKV from complex environments.

SARS-Cov2.
The COVID-19 caused the global spreads and immeasurable damages. And more than six million patients was confirmed to be infected with SARS-Cov2 until Jun-4-2020. The high mortality brought unprecedented influence on human society. Thus, the monitoring of highly pathogenic virus is of great significance to the prevention and control of emerging infectious disease. In this section, the sequence of S protein and N protein gene were chosen as the instances for detection of SARS-Cov2 with dendritic Ru(bpy)3 2+ -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 dendritic Ru(bpy)3 2+ -polymer amplified ECL-scan imaging system for SARS-Cov2. The results in Figure 7B and C indicated that dendritic Ru(bpy)3 2+ -polymer amplified ECL-scan imaging system achieved the 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 Figure S8 and S9. The detections of spiked saliva and blood samples also conformed the outstanding 15 capacity of dendritic Ru(bpy)3 2+ -polymer amplified ECL-scan imaging system for detection human samples.

Conclusions
A dendritic polymer probe-amplified ECL-scan imaging system was constructed to realize trace analysis of viral emerging infectious disease. Dendritic polymer probe was employed as the efficient signal giving-out component that could generate 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 were 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 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 virus, and thus has the potential to serve as a liquid biopsy paradigm for monitoring of viral emerging infectious disease.

Supplementary Information
The online version contains supplementary material available at

Availability of data and materials
All data generated or analyzed during this study are included in this published article and its additional file 1.

Ethics approval and consent to participate
All procedures involving experimental animals and clinical samples were carried out under guidelines approved by the Ethics Committee of Southern Medical University.