key: cord-0693717-ts0vj0k9
authors: Fan, Zhenqiang; Yao, Bo; Ding, Yuedi; Xie, Minhao; Zhao, Jianfeng; Zhang, Kai; Huang, Wei
title: Electrochemiluminescence aptasensor for Siglec-5 detection based on MoS(2)@Au nanocomposites emitter and exonuclease III-powered DNA walker
date: 2021-02-08
journal: Sens Actuators B Chem
DOI: 10.1016/j.snb.2021.129592
sha: c313ad436ee12c3d02369a0b0153e16798d9157a
doc_id: 693717
cord_uid: ts0vj0k9

Lectins are highly specific binding proteins for glycoproteins which widely exist in living organisms, playing a vital role in exploring the biological evolution process, such as cellular proliferation, differentiation, carcinogenesis and apoptosis. Therefore, the content monitoring of lectin becomes particularly significant and urgent in the bioanalytical application. In this work, we fabricated an aptasensor, majorly capitalizing the eminent affinity between sialic acid-binding immunoglobulin (Ig)-like lectin 5 (Siglec-5) and nucleic acids aptamer (K19), with nontoxic MoS(2)@Au nanocomposites as electrochemiluminescence (ECL) emitters based on exonuclease III (Exo III)-powered DNA walker for the bioassays of Siglec-5. The DNA track was constructed on the emitters’ surface, providing a reliable platform for the DNA walker’s autonomous move. In the assay, the primer DNA in the DNA duplex was replaced by Siglec-5 due to the aptamer interactions and repeatedly released to participate in the movement of the DNA walker, further triggering cascade signal amplification. Finally, our aptasensor indicates significant potential for assays of Siglec-5 with a detection limit of 8.9 pM.

The raging of 2019 novel coronavirus (2019-nCoV) with a rapid transmission spread in the worldwide area, had caused severe effects on travel, life and work of hundreds of millions to an unprecedented level in the past year [1, 2] . However, no valid vaccines or therapeutic drugs were widely applied for the treatment of this highly contagious coronaviruses by now [3] . The three types of proteins encoded by the 2019-nCoV genome, including non-structural proteins, structural proteins, and accessory proteins are envisaged as potential targets for the development of human coronavirus antiviral agents [4] . The study of spike glycoprotein, an important structural protein that plays a crucial role in the interaction between viruses and cellular receptors, is beneficial for understanding the infection process of virus entry into cells and for obtaining new interventions [5] . Lectins are highly specific binding proteins for various viral surface glycoproteins, and the evaluation of lectin efficacy and the delivery route provides a potential short-term strategy for the development of coronavirus drugs at the molecular scale [6] . Sialic acid-binding immunoglobulin (Ig)-like lectins (Siglecs) as a member of the lectin family, the significance and quantitative analysis of which also need further evaluation and accurate implementation.

Siglecs are cell-surface receptors belonging to the immunoglobulin superfamily and each cell surface receptor consists of 2-17 extracellular Ig domains that contain the sialic acid-binding sites [7] [8] [9] . These type-I transmembrane proteins are known to us for recognizing negatively charged sialic acids linked to the terminal of non-reducing end of most glycans on scaffolds of proteoglycans, glycosphingolipids, and glycoproteins [10] . They are predominantly expressed in hematopoietic cells and participates in the modulation of physiological cellular processes by a host of glycanprotein interactions [10] .

In terms of sequence homology, the Siglecs family can be split into two groups. The

Siglecs known as CD33-related contain Siglec-3 (CD33), Siglec-5 (CD170), Siglec-11

and Siglec-14, which show high sequence homology (~50-99% sequence identity) in the Siglecs subfamily [11] . The other category includes Siglec-1, Siglec-2 (CD22), Siglec-4 and Siglec-15 with low sequence homology (25-50% sequence identity) [12, 13] . As one of the most common Siglecs, Siglec-5 has serval-linked conformations (α2-3, α2-6, α2-8) to recognize specific sialic acids and is selectively expressed in human monocytes, neutrophils, basophils and dendritic cells [14] . Interestingly, this type of Siglec consists of two intracellular "immunoreceptor tyrosine-based inhibitory motifs" (ITIM) that prevent kinase-mediated activation responses and are utilized as inhibitory receptors for leukocytes [10, 15] . For these advantages, Siglec-5 has been regarded as the disease-specific biomarker and possible target protein of scores of immune-related diseases such as primary Sjogren's syndrome (pSS) and acute myeloid leukemia (AML) [16] . To improve the survival rates of these diseases, novel methods with high sensitivity and selectivity to discover Siglec-5 for the assays and targeted therapy are needed.

Aptamer-based technology as a distinct strategy in support of individualized medicine has exhibited extraordinary potential, partly bringing about the identification and J o u r n a l P r e -p r o o f validation of molecular signatures [17] [18] [19] . However, the discoveries of currently available aptamers of the biomarkers and valid analytical platforms are becoming imperative and valuable in bioanalysis and therapeutics. Systematic evolution of ligands by exponential enrichment (SELEX) as a molecular tool to screen nucleic acids from a library of random single-stranded nucleic acid sequences, with specific affinity to target proteins which is commensurate with individual antibodies, provides an effective means for biomarker investigations [20] [21] [22] . Based on SELEX selection technology, aptamer K19 with equilibrium dissociation constants (Kd) of 12.37 nM for NB4 AML cells shows high affinity for Siglec-5 which is regarded as the biomarker of AML and target protein of the K19 [23] . Additionally, the construct of the aptamerbased platform is envisioned to realize a quantitative approach for biomarker discovery and further leukemic therapy.

The aptamer-based analysis platforms are typically fabricated based on fluorescence (FL), electrochemistry (EC), and electrochemiluminescence (ECL), which are widely used in the areas of bioanalysis and clinical diagnosis [24] [25] [26] . ECL as a unique analytical technique is so charming for its characteristics of the low-cost device, simple operation and high sensitivity, avoiding the shortcoming of fluorescence using external background light and combining the advantage of electrochemistry with high sensitivity [27, 28] . Besides, electrochemiluminescence-resonance energy transfer (ECL-RET) means the energy transfer between the adjacent emitters and the receptors, which was widely utilized in the design of biosensors and provided an efficient method for the detection of tumor markers [29, 30] . In the last few years, ECL emitters ranged from traditional Ru(bpy)3 2+ to newly emerged materials such as Cd-based quantum dots (QDs), metal complexes, organic nanocrystals, and a series of high quantum efficiency materials [27, [31] [32] [33] [34] . MoS2 QDs as classical graphene-analogous transition metal twodimensional layered sulfide with a size-tunable bandgap, possess unique electronic characteristics, large specific area and heavy-metal-free properties, which have great applicable potential in multiphoton bioimaging, electrocatalytic activity as well as in J o u r n a l P r e -p r o o f ECL behavior [35, 36] . Therefore, MoS2 QDs as biocompatible ECL emitters have the advantages of lower toxic metal ions and excellent characteristics compared to traditional Cd-based QDs [37] . However, limited by the intrinsic conductivity and the limited surface modification groups, the application of MoS2 QDs on the electrode surface for biosensors is limited [38] . In order to improve such conditions, compounding with gold nanoparticles provides an efficient approach which supplies a huge area for MoS2 QDs to attach, and facilitates the immobilization of nucleotide through the formation of Au-S bond, further promoting the construction of the sensor [39] .

In the past decades, nucleic acid nanotechnology has supplied enormous possibilities for the operation of biochemical engineering [40] . In particular, dynamic nucleic acids nanotechnology is applied in artificial intelligence areas including smart biosensors, intelligent computing, nimble disease diagnosis and drug delivery [41] [42] [43] . One type of dynamic aspects is the DNA walker, and the foremost property of which is the performance in autonomous and highly directional mechanical movement in linear, planar and three-dimensional tracks through dynamic interactions [44] . DNA walker has been widely used in the fabrication of biosensors for the assay of target DNA, miRNA and bifunctional proteins. For example, Chai et al. utilized a bipedal DNA walker-based electrochemical strategy for the assays of specific sequences [45] . Peng et al. realized the analysis of miRNA using a bi-directional DNA machine and enzyme-free strategy [46] . Jiang et al. fabricated DNA nanomachine-based ECL biosensor for the detection of Mucin 1 [47] . The application of a DNA walker in the ultra-micro bioanalysis supplies reliable methods to ameliorate the bioanalytical platforms and improve diagnostic efficiency.

Herein, an electrochemiluminescence aptasensor with high sensitivity, rapid operation and simple device, using MoS2@Au nanocomposites as efficient emitter and Exo IIIpowered DNA walker for isothermal signal amplification, was fabricated for sensitive detection of Siglec-5. This strategy was based on ECL-RET, of which MoS2@Au J o u r n a l P r e -p r o o f nanocomposites as ECL emitter and the gold nanocage served as a quencher, for the reason that the absorbance of Au nanocages was overlapped with the ECL spectra of MoS2@Au nanocomposites, which significantly improved the quenching efficiency of the quenchers. Compared to gold nanoparticles or nanoshells, the absorbance spectra of Au nanocages had more overlapping parts with the ECL spectra of MoS2@Au nanocomposites [48, 49] . In the presence of target Siglec-5, the multifunctional doublestranded DNA (dsDNA) probe formed by aptamer and primer DNA was disintegrated, and the primer DNA was released to further hybridize with the Au nanocages-labeled stator DNA (S2) probe already partially hybridized with staple DNA (S1). With the help of unidirectional digestion of Exo III, movement of single foot DNA walker on the MoS2@Au nanocomposites was conducted. The DNA nanomachine supplied an efficient way to transduce and amplify recognition signals. Most importantly, due to the excellent ECL emission of MoS2@Au nanocomposites, high-performance quenching platform and Exo III-powered signal amplification strategy using stochastic DNA walker, we had successfully fabricated an aptasensor for detecting Siglec-5 with high sensitivity and excellent selectivity.

Regents and chemicals, unless otherwise stated, were got from Sangon Biotech Co. Ltd. Table S1 . The water used in this strategy was purified with resistance 5 MΩ using a Milli-Q purification system (Branstead).

Scanning electron microscopy (SEM) images and energy-dispersive X-ray (EDX) were from JSM-7800F (Japan). ECL tests were carried out using ECL-6B equipped with a 

Firstly, molybdenum disulfide powders were pretreated to get a well-dispersed MoS2

QDs solution according to already published literatures [50, 51] . In short, 300 mg molybdenum disulfide was firstly dissolved in 30 mL N, N-dimethylformamide (DMF).

Subsequently, continuous sonication was performed for 6 h, and the obtained mixture was refluxed at 140 ℃ for 8 h. After that, the sonication of the resultant solution was conducted for 2.5 h and followed by centrifugation at 5000 rpm to get a pale yellow supernatant. Next, the supernatant was evaporated under vacuum to remove redundant DMF, and then deionized water was added to get MoS2 QDs solution.

Then, 2 mL MoS2 QDs aqueous solution was added to 4 mL 50-nm-sized gold colloid solution and slowly heated to 50 ℃ under stirring for 8 h. Besides, centrifugation at 10000 rpm was performed to obtain MoS2@Au nanocomposites. Finally, the MoS2@Au nanocomposites were stored in the dark at 4 ℃ for the following utilization.

The synthesis of DNA-Au nanocages composites was referring to previous literature with slight modifications [52] . In short, 1 mL newly purchased 20-nm-sized nanocages solution (2 nM) with metallic luster and polyhedral structure (the SEM characterization was in Fig. S1 ) containing 200 nM mPEG-SH with a molecular weight of 5 kDa was mixed with 10 μL (1 wt%) Tween 20. After shaking the mixture for 3 min, 10 μL (100 μM) deprotected thiol-terminated stator DNA was put together and NaCl aqueous solution was added with a final concentration of 500 mM. Left at room temperature for 1 h, the mixture was centrifugated at 10000 rpm. After being repeatedly washed three times, the precipitate was redispersed in 1 mL PBS (0.1 M) and put in the refrigeration at 4 ℃ for further modification.

Initially, a glassy carbon electrode with a diameter of 4 mm was constantly polished using 0.05 μm alumina, followed by sonication in deionized water to get a mirror electrode. After that, 10 μL prepared MoS2@Au solution was dropped onto the spotless GCE and dried for 1 h to form MoS2@Au modified electrode (abbreviation as For every modification of the electrode, PBS (100 mM, pH 7.1) was used to rinse out the non-specifically substances. As for endogenous assessment in cell nuclear extract samples, the process of cell culture and the preparation of nuclear extract were referred to our previous work [53] . ECL signals were obtained through PBS containing 20 mM triethylamine with the voltage scanning range from 0 to 1.3 V. Exo III using exonuclease activity. The Exo III exclusively cleaved nucleotides from blunt or concave 3' termini of dsDNA but expressed no activity on ssDNA or convex 3' termini (more than four bases) of dsDNA [54] . Therefore, the sequence of stator DNA paired with staple DNA was merely digested, yielding a 6-nt oligo carrying Au nanocage, but the consumption of mononucleotides of primer DNA was blocked by extended sequences (black extensions) in 3' termini. The released primer DNA then hybridized with another strand of stator DNA through toehold-mediated branch migration and triggered a new digestion activity, which tended to form automatically cyclical motions reaching cognate clarity as isothermal amplification reactions. As the walking process proceeded, the quencher was gradually removed from the stator DNA and the "signal on" state reappeared. 

For the verification of the assay process of Siglec-5, we employed polyacrylamide gel electrophoresis (PAGE) to characterize the bioanalytical process, and the PAGE image was depicted in Fig. 2A respectively. Three strips in lane 4 corresponded to the primer, aptamer and its duplex DNA probe, respectively. Therefore, we could infer that Siglec-5 competed with primer DNA for the hybridization with K19 aptamer, and thus some primer DNA probe was released. The band representing aptamer DNA indicated that the binding of aptamer and Siglec-5 was undermined during the PAGE process. The three bands in lane 7 contained two strips with the same migration speeds as the primer and the staple DNA as well as a newly emerged strip, revealing that with Exo III the sequences of stator DNA were digested and primer DNA probe released with high concentration. Also, the dark stripe with the lowest migration speed should be speculated as the residual hybridization strands of staple, stator and primer DNA probes during the walker process.

For further systematically investigating the hydrolysis effects of different enzyme systems, for instance, the effects of DNase I, Exo I, and Exo III on nucleotides in this DNA walker were also explored (Fig. 2B) . DNase I as an endonuclease could digest ssDNA and dsDNA randomly. Exo I and Exo III exclusively digested ssDNA and dsDNA in 3' to 5' direction, severally. The hydrolysis reactions were monitored according to the ECL response of the three enzymatic reactions under significant changes of Siglec-5 concentrations, showing that the ECL intensity enhanced along with the separation of ECL emitter and quencher (Au nanocages). As for DNase I, J o u r n a l P r e -p r o o f hydrolysis degree was the highest in the three types of nucleases, while did not change with the target protein concentration. Exo I showed a generally similar result that hydrolysis reaction had nothing to do with the target protein and the separation of emitter and quencher was blocked due to its cleaving characteristics. The hydrolysis efficiency of Exo III was significantly influenced by the target concentration due to its high participatory for DNA walker, which indicated the importance of Exo III for the assay of Siglec-5 of this aptasensor.

ECL and electrochemical behaviors of MoS2 QDs and MoS2 QDs modified Au nanoparticles were investigated to certificate the emitting mechanism of the emitter. As shown in Fig. 3A , a feeble ECL signal of MoS2 QDs with approximately 50 a.u. was observed (curve a) in PBS. Meanwhile, its corresponding CV testing as depicted in Fig.   3B , indicated that the maximum current was slight (curve a). However, when TEA was added to the MoS2-based ECL system, the ECL intensity and maximum current were extremely raised, with ECL intensity increased to 910 a.u. (curve b in Fig. 3A) , about 18.7 folds compared to the ECL system without co-reactant TEA, and maximum current dramatically enhanced to 325 μA (curve b in Fig. 3B ). Besides, in the working buffer containing TEA, MoS2@Au nanocomposites exhibited more obvious enhancement compared to MoS2 QDs in ECL and electrochemical aspects, the ECL intensity of which increased to about 1311 a.u. (curve c in Fig. 3A) , and the maximum current was heightened to 381 μA (curve c in Fig. 3B ). We inferred that the Au nanoparticles here acted as the catalyst to improve the efficiency of charge transfer and reinforced ECL emission. One possible ECL mechanism of MoS2 QDs could be illustrated as below. In brief, TEA lost an electron to be oxidized to TEA• with strong reduction and participate to the ECL process of reducing MoS2 QDs for the generation of the electronically excited state of MoS2 * . At last, MoS2 * released energy (light) and back to ground state. 

We applied CV, Nyquist plot of impedance spectra and ECL emission for the characterization of the step-by-step modifications of aptasensor on the electrode. As In this stepwise modification, Rct was driven up from a to d, which denoted the impedance between the working electrode, and the reference electrode was gradually increasing. With the modification of stator DNA-Au nanocages (curve e), Rct decreased slightly for that nanocages enhanced the efficiency of electron transfer and reduced the interfacial resistance. After the process of DNA walker (curve f), the Rct raised again for the reason that the nanocages were set free.

Besides, ECL response (Fig. 4C) with good sensitivity and suitable for Siglec-5 analysis [16] . For further exploring the detection performance, the recovery rate was also shown in Table 1 . The recovery rates of data 1 presented a narrow range (96.1%-103.68%) when detecting spiked Siglec-5

in Tris-HCl solution, expressing that this method possessed good feasibility. The endogenous Siglec-5 was measured to test the practical value of this method.

Specifically, Siglec-5 in 10-fold diluted cell nuclear extracts was detected for the assessment of endogenous influences of other components in the actual detecting environment. As shown in Table 1 

In addition, to verify the application prospects of the proposed aptasensor, we explored the reproducibility of the ECL signal by performing 10 consecutive scans of the cyclic potential at three target concentrations (100, 200 and 300 pM) as shown in Fig. 5C . The lower relative standard deviations (RSD) denoted that its repeatability was within the acceptable range (<5%). Since selectivity was of importance for aptasensor, further researches on it were conducted. Extra nonspecific proteins, for example, bovine serum albumin (BSA), NF-κB p50, human serum albumin (HSA), interleukin-6 (IL-6) and

their mixture were selected to compare with Siglec-5 in the sensing assays (Fig. 5D ).

Besides, we selected a variety of other types of lectins, including galectin-3, Siglec-8, and snailagglutinin for investigating the resistance of the biosensor system to these interfering lectin proteins to highlight the specificity of the system for the specifically J o u r n a l P r e -p r o o f Siglec-5 (Fig. S8) . These results revealed an unignorable disparity that the ECL signal enhancement for target protein was higher than that for 10-fold excess of other proteins, indicating the aptasensor with excellent selectivity for Siglec-5.

Related lectins with binding properties to spike glycoproteins are potentially valuable for the developing antiviral agents against 2019-nCoV. Thus, rapid identification and quantitative assays of lectins are necessary for the early discovery and analysis of coronaviruses. In our work, an ECL aptasensor using MoS2@Au nanocomposites as emitters and Exo III-propelled DNA walker as a signal amplification strategy was constructed for quantitative bioanalysis of Siglec-5. Employing the high binding affinity between Siglec-5 with K19 aptamer, we ingeniously transformed the assays of target protein into the assays of primer DNA, which further participated in the motion of the DNA walker for signal amplification. Besides, we constructed a notable DNA walking track on MoS2@Au nanocomposites and caused a "signal off" state, which with the DNA walker process gone ahead, the state reversed to "signal on". Finally, our ECL system exhibited strong anti-interference ability with a superior selectivity for ultrasensitive assay of Siglec-5, performing a detection limit as low as 8.9 pM and providing a feasible analysis method for Siglec-5 in a single A549 cell, which possessed huge potential in evaluating the role of lectins at a cellular level and developing anticoronavirus agents in 2019 novel coronaviruses epidemic.

Supplementary data can be found in the online version.

Credit Author Statement 

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 

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Biographies Zhenqiang Fan works on electrochemical based biosensors in Jiangsu Institute of Nuclear Medicine

Yao Bo is a master student at Nanjing Tech University, China. His main research interests are biosensors

Yuedi Ding's field and main subject of current research interests are the application of biosensors in single-cell assay

Minhao Xie received his Ph.D. degree at Jiangnan University, China. He currently works as a professor at Jiangsu Institute of Nuclear Medicine

He received a Ph.D. degree from Nanjing University of Posts and Telecommunications in 2011. His main research interests are micro/nano materials and chemical sensors

Zhang currently works as an associate professor at Jiangsu Institute of Nuclear Medicine. His current research interests include nanostructured functional materials and their application for chemical and biosensors

He is a member of the Chinese Academy of Sciences and also a foreign member of the Russian Academy of Sciences, as well ASA member of the Asian Pacific Academy of Materials

This work was supported by the National Natural Science Foundation of China 

for the assays of Siglec-5 using Exo III-driven DNA walkers.