Publications by authors named "Xiaoyong Zou"

67 Publications

Highly Sensitive Fluorescence Detection of Global 5-Hydroxymethylcytosine from Nanogram Input with Strongly Emitting Copper Nanotags.

Anal Chem 2021 Oct 12. Epub 2021 Oct 12.

School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, China.

Quantitative analysis of 5-hydroxymethylcytosine (5hmC) has remarkable clinical significance to early cancer diagnosis; however, it is limited by the requirement in current assays for large amounts of starting material and expensive instruments requring expertise. Herein, we present a highly sensitive fluorescence method, termed hmC-TACN, for global 5hmC quantification from several nanogram inputs based on terminal deoxynucleotide transferase (TdT)-assisted formation of fluorescent copper (Cu) nanotags. In this method, 5hmC is labeled with click tags by T4 phage β-glucosyltransferase (β-GT) and cross-linked with a random DNA primer via click chemistry. TdT initiates the template-free extension along the primer at the modified 5hmC site and then generates a long polythymine (T) tail, which can template the production of strongly emitting Cu nanoparticles (CuNPs). Consequently, an intensely fluorescent tag containing numerous CuNPs can be labeled onto the 5hmC site, providing the sensitive quantification of 5hmC with a limit of detection (LOD) as low as 0.021% of total nucleotides (/ = 3). With only a 5 ng input (∼1000 cells) of genomic DNA, global 5hmC levels were accurately determined in mouse tissues, human cell lines (including normal and cancer cells of breast, lung, and liver), and urines of a bladder cancer patient and healthy control. Moreover, as few as 100 cells can also be distinguished between normal and cancer cells. The hmC-TACN method has great promise of being cost effective and easily mastered, with low-input clinical utility, and even for the microzone analysis of tumor models.
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http://dx.doi.org/10.1021/acs.analchem.1c03266DOI Listing
October 2021

A MOF-Shell-Confined I-Motif-Based pH Probe (MOFC-i) Strategy for Sensitive and Dynamic Imaging of Cell Surface pH.

ACS Appl Mater Interfaces 2021 Sep 20;13(38):45291-45299. Epub 2021 Sep 20.

School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, China.

Dynamic imaging of cell surface pH is extremely challenging due to the slight changes in pH and the fast diffusion of secreted acid to the extracellular environment. In this work, we construct a novel metal-organic framework (MOF)-shell-confined i-motif-based pH probe (MOFC-i) strategy that enables sensitive and dynamic imaging of cell surface pH. The CY3- and CY5-labeled i-motif, which is hybridized via its short complementary chain with two-base mismatches, is optimized for sensing at physiological pH. After efficiently anchoring the optimized pH probes onto the cell membrane with the aid of cholesterol groups, a biocompatible microporous MOF shell is then formed around the cell by cross-linking ZIF-8 nanoparticles via tannic acid. The microporous MOF shell can confine secreted acid without inhibiting the normal physiological activities of cells; thus, the MOFC-i strategy can be used to monitor dynamic changes in the cell surface pH of living cells. Furthermore, this method can not only clearly distinguish the different metabolic behaviors of cancer cells and normal cells but also reveal drug effects on the cell surface pH or metabolism, providing promising prospects in pH-related diagnostics and drug screening.
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http://dx.doi.org/10.1021/acsami.1c13720DOI Listing
September 2021

Isothermal Self-Primer EXPonential Amplification Reaction (SPEXPAR) for Highly Sensitive Detection of Single-Stranded Nucleic Acids and Proteins.

Anal Chem 2021 09 7;93(37):12707-12713. Epub 2021 Sep 7.

School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, P. R. China.

Development of versatile sensing methods for sensitive and specific detection of clinically relevant nucleic acids and proteins is of great value for disease monitoring and diagnosis. In this work, we propose a novel isothermal Self-primer EXPonential Amplification Reaction (SPEXPAR) strategy based on a rationally engineered structure-switchable Metastable Hairpin template (MH-template). The MH-template initially keeps inactive with its self-primer overhanging a part of target recognition region to inhibit polymerization. The present targets can specifically compel the MH-template to transform into an "activate" conformation that primes a target-recyclable EXPAR. The method is simple and sensitive, can accurately and facilely detect long-chain single-stranded nucleic acids or proteins without the need of exogenous primer probes, and has a high amplification efficiency theoretically more than 2. For a proof-of-concept demonstration, the SPEXPAR method was used to sensitively detect the characteristic sequence of the typical swine fever virus (CSFV) RNA and thrombin, as nucleic acid and protein models, with limits of detection down to 43 aM and 39 fM, respectively, and even the CSFV RNA in attenuated vaccine samples and thrombin in diluted serum samples. The SPEXPAR method may serve as a powerful technique for the biological research of single-stranded nucleic acids and proteins.
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http://dx.doi.org/10.1021/acs.analchem.1c02588DOI Listing
September 2021

Cancer cell identification by facile imaging of intracellular reductive substances with fluorescent nanosensor.

Talanta 2021 Nov 25;234:122650. Epub 2021 Jun 25.

School of Chemistry, Sun Yat-Sen University, Guangzhou, 510275, China. Electronic address:

Ascorbic acid (AA) and glutathione (GSH), the most abundant intracellular reductive substances, have been widely used as biomarkers for cancer cells identification. The current methods relying on imaging of AA or GSH alone to identify cancer cells may cause systematic errors, since a mutual conversion relationship exists between AA and GSH. In this work, we propose a fluorescent nanosensor for the simultaneous imaging of intracellular reductive substances including AA and GSH. Biocompatible fluorescent silicon nanoparticles (SiNPs) with rich surface amine and carboxyl groups were synthesized. The fluorescence of the SiNP was initially quenched by chelation of Fe ions, forming SiNP/Fe complex as the fluorescent nanosensor. Upon the redox reaction with reductive substances, the nanosensor showed sensitively fluorescent recovery. Moreover, benefited from the efficient cellular uptake of the SiNP/Fe and the overexpressed intracellular reductive substances in cancer cells, the fluorescent nanosensor was used to accurately identify the human breast carcinoma (MCF-7) cells from normal mammary epithelial (MCF-10A) cells by imaging of intracellular AA and GSH simultaneously. This strategy would be promising in imaging-guided precision cancer diagnosis.
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http://dx.doi.org/10.1016/j.talanta.2021.122650DOI Listing
November 2021

Pearl Necklacelike Strategy Enables Quantification of Global 5-Hydroxymethylcytosine and 5-Formylcytosine by Inductively Coupled Plasma-Atomic Emission Spectrometry.

Anal Chem 2021 06 26;93(22):7787-7791. Epub 2021 May 26.

School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, China.

5-Hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) are key intermediates of active DNA demethylation, for which the global detection methods are still restricted by high cost and long operation time. Here, we demonstrate a pearl necklacelike strategy to accurately quantify global 5hmC and 5fC in genomic DNA. In this method, the metal-organic framework (MOF), [Cu(BTC)] (denoted as HKUST-1, HBTC = 1,3,5-benzenetricarboxylic acid), with a diameter of ∼30 nm that contains ∼15 000 copper ions (Cu) as the "super label" was grown in the carboxylated 5hmC and 5fC loci of genomic DNA via the coordination between Cu and the carboxyl group. After the acid digestion of MOF, the concentration of Cu, which has a quantitative relationship with the 5hmC/5fC content, was measured by inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The metal element enrichment during MOF growth has amplified the signal by 4 orders of magnitude, realizing sensitive and accurate quantification of global 5hmC and 5fC in different tissues with a detection limit of 0.031% and 0.019‰ in DNA, respectively. The bisulfite- and mass spectrometry-free strategy is easily performed in almost all research and medical laboratories and would provide potential capability to quantify other candidate modifications in nucleotides.
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http://dx.doi.org/10.1021/acs.analchem.1c01548DOI Listing
June 2021

Novel Potential Small Molecule-MiRNA-Cancer Associations Prediction Model Based on Fingerprint, Sequence, and Clinical Symptoms.

J Chem Inf Model 2021 05 26;61(5):2208-2219. Epub 2021 Apr 26.

School of Chemistry and Chemical Engineering, Guangdong Pharmaceutical University, Guangzhou 510006, People's Republic of China.

As an important biomarker in organisms, miRNA is closely related to various small molecules and diseases. Research on small molecule-miRNA-cancer associations is helpful for the development of cancer treatment drugs and the discovery of pathogenesis. It is very urgent to develop theoretical methods for identifying potential small molecular-miRNA-cancer associations, because experimental approaches are usually time-consuming, laborious, and expensive. To overcome this problem, we developed a new computational method, in which features derived from structure, sequence, and symptoms were utilized to characterize small molecule, miRNA, and cancer, respectively. A feature vector was construct to characterize small molecule-miRNA-cancer association by concatenating these features, and a random forest algorithm was utilized to construct a model for recognizing potential association. Based on the 5-fold cross-validation and benchmark data set, the model achieved an accuracy of 93.20 ± 0.52%, a precision of 93.22 ± 0.51%, a recall of 93.20 ± 0.53%, and an F1-measure of 93.20 ± 0.52%. The areas under the receiver operating characteristic curve and precision recall curve were 0.9873 and 0.9870. The real prediction ability and application performance of the developed method have also been further evaluated and verified through an independent data set test and case study. Some potential small molecules and miRNAs related to cancer have been identified and are worthy of further experimental research. It is anticipated that our model could be regarded as a useful high-throughput virtual screening tool for drug research and development. All source codes can be downloaded from https://github.com/LeeKamlong/Multi-class-SMMCA.
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http://dx.doi.org/10.1021/acs.jcim.0c01458DOI Listing
May 2021

Toehold-mediated ligation-free rolling circle amplification enables sensitive and rapid imaging of messenger RNAs in situ in cells.

Anal Chim Acta 2021 May 29;1160:338463. Epub 2021 Mar 29.

School of Chemistry, Sun Yat-Sen University, 135 Xingang West Road, Guangzhou, 510275, PR China.

In situ analysis of tumor-related messenger RNAs (mRNAs) is significant in identifying cancer cells at the genetic level in the early stage. Rolling circle amplification (RCA)-based methods are primary tools for in situ mRNA assay, however, the necessary ligation reaction not only shows low ligation efficiency, but also greatly prolongs the assay time that increases the risk of cells losing and mRNAs leakage. In this work, we propose a novel toehold-mediated ligation-free RCA (TMLFRCA) on a designed structure-switchable dumbbell-shaped probe (SDP). Target mRNA can specifically activate SDP from its circular form by toehold strand displacement, thereby initiates in situ RCA for mRNA imaging with the help of a short DNA primer. For the proof-of-concept demonstration, the TK1 mRNA was sensitively detected by TMLFRCA in less than 3.5 h with a limit of detection (LOD) of 0.39 fM (corresponds to 2.39×10copiesL), and significantly improved specificity capable for distinguishing single base difference. The sensitivity of the TMLFRCA for TK1 mRNA in situ assay is ∼29-fold and ∼7-fold higher than that of FISH and ligase-assisted RCA method, respectively, which enables the TMLFRCA method capability of highly sensitive and specific distinction mRNA expression levels between cancer cells and normal cells. We believe this TMLFRCA strategy would be of great value in both basic research and clinical diagnosis.
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http://dx.doi.org/10.1016/j.aca.2021.338463DOI Listing
May 2021

Performance improvement for a 2D convolutional neural network by using SSC encoding on protein-protein interaction tasks.

BMC Bioinformatics 2021 Apr 12;22(1):184. Epub 2021 Apr 12.

School of Chemistry, Sun Yat-Sen University, Guangzhou, 510275, People's Republic of China.

Background: The interactions of proteins are determined by their sequences and affect the regulation of the cell cycle, signal transduction and metabolism, which is of extraordinary significance to modern proteomics research. Despite advances in experimental technology, it is still expensive, laborious, and time-consuming to determine protein-protein interactions (PPIs), and there is a strong demand for effective bioinformatics approaches to identify potential PPIs. Considering the large amount of PPI data, a high-performance processor can be utilized to enhance the capability of the deep learning method and directly predict protein sequences.

Results: We propose the Sequence-Statistics-Content protein sequence encoding format (SSC) based on information extraction from the original sequence for further performance improvement of the convolutional neural network. The original protein sequences are encoded in the three-channel format by introducing statistical information (the second channel) and bigram encoding information (the third channel), which can increase the unique sequence features to enhance the performance of the deep learning model. On predicting protein-protein interaction tasks, the results using the 2D convolutional neural network (2D CNN) with the SSC encoding method are better than those of the 1D CNN with one hot encoding. The independent validation of new interactions from the HIPPIE database (version 2.1 published on July 18, 2017) and the validation of directly predicted results by applying a molecular docking tool indicate the effectiveness of the proposed protein encoding improvement in the CNN model.

Conclusion: The proposed protein sequence encoding method is efficient at improving the capability of the CNN model on protein sequence-related tasks and may also be effective at enhancing the capability of other machine learning or deep learning methods. Prediction accuracy and molecular docking validation showed considerable improvement compared to the existing hot encoding method, indicating that the SSC encoding method may be useful for analyzing protein sequence-related tasks. The source code of the proposed methods is freely available for academic research at https://github.com/wangy496/SSC-format/ .
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http://dx.doi.org/10.1186/s12859-021-04111-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8042949PMC
April 2021

Fluorescence on-off-on with small and charge-tunable nanoparticles enables highly sensitive intracellular microRNA imaging in living cells.

Talanta 2021 May 15;226:122114. Epub 2021 Jan 15.

School of Chemistry, Sun Yat-Sen University, Guangzhou, 510275, China. Electronic address:

Nanomaterial-based on-off-on fluorescence sensing strategies are significant particularly in intracellular nucleic acids imaging assay. There still remains challenge to rationally balance fluorescence quenching efficiency and recovery dynamics. We assume that the performance of on-off-on fluorescence sensing strategy can be fundamentally improved on small zero-dimensional (0D) nanomaterial with precisely modulated surface charge. For a proof-of-concept demonstration, silicon nanoparticle (SiNP) with ~4 nm was synthesized and used as the quencher model, of which the surface charge density was modulated by modification of triphenylphosphonium (TPP). The influence of particle size, surface charge and charge density of the nanomaterials on sensing performance was systematically investigated. The strategy showed a low limit of detection (LOD) as 26 pM for target model miR-494, which is one of the lowest in nanomaterial-based on-off-on sensing platforms. And the LOD is even comparable to amplification-based methods in a greatly shortened assay time (2.5 h). The miR-494 expresses in cancerous and normal living cells of human cervical carcinoma (HeLa), human lung carcinoma (A549), human breast cancer (MCF-7), and normal human mammary epithelial (MCF-10A) cells were imaged and localized with significantly improved sensitivity and specificity. These excellent performances insure it a promising candidate as convenient and non-enzymatic sensing platform for miRNA-associated disease detection and early diagnosis.
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http://dx.doi.org/10.1016/j.talanta.2021.122114DOI Listing
May 2021

A protein triggering exponential amplification reaction enables label- and wash-free one-pot protein assay with high sensitivity.

Talanta 2021 Apr 9;225:121980. Epub 2020 Dec 9.

School of Chemistry, Sun Yat-Sen University, Guangzhou, 510275, PR China.

Methods capable of sensitive and facile quantification of low-abundant proteins play critical roles in disease diagnosis and treatment. Herein, on a rationally designed aptamer-based hairpin structure-switching template, we developed a protein triggering exponential amplification reaction (PTEXPAR) method. The platelet-derived growth factor BB (PDGF-BB) is used as model analyte in the current proof-of-concept experiments. This method can detect PDGF-BB specifically with a detection limit as low as 4.9 fM. Additionally, the proposed PTEXPAR strategy allows label- and wash-free one-pot quantification of protein within ~35 min. Moreover, it is potentially universal because hairpin template can be easily designed for other proteins by changing the corresponding aptamer sequence.
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http://dx.doi.org/10.1016/j.talanta.2020.121980DOI Listing
April 2021

Seq-SymRF: a random forest model predicts potential miRNA-disease associations based on information of sequences and clinical symptoms.

Sci Rep 2020 10 21;10(1):17901. Epub 2020 Oct 21.

School of Chemistry and Chemical Engineering, Guangdong Pharmaceutical University, Guangzhou, 510006, People's Republic of China.

Increasing evidence indicates that miRNAs play a vital role in biological processes and are closely related to various human diseases. Research on miRNA-disease associations is helpful not only for disease prevention, diagnosis and treatment, but also for new drug identification and lead compound discovery. A novel sequence- and symptom-based random forest algorithm model (Seq-SymRF) was developed to identify potential associations between miRNA and disease. Features derived from sequence information and clinical symptoms were utilized to characterize miRNA and disease, respectively. Moreover, the clustering method by calculating the Euclidean distance was adopted to construct reliable negative samples. Based on the fivefold cross-validation, Seq-SymRF achieved the accuracy of 98.00%, specificity of 99.43%, sensitivity of 96.58%, precision of 99.40% and Matthews correlation coefficient of 0.9604, respectively. The areas under the receiver operating characteristic curve and precision recall curve were 0.9967 and 0.9975, respectively. Additionally, case studies were implemented with leukemia, breast neoplasms and hsa-mir-21. Most of the top-25 predicted disease-related miRNAs (19/25 for leukemia; 20/25 for breast neoplasms) and 15 of top-25 predicted miRNA-related diseases were verified by literature and dbDEMC database. It is anticipated that Seq-SymRF could be regarded as a powerful high-throughput virtual screening tool for drug research and development. All source codes can be downloaded from https://github.com/LeeKamlong/Seq-SymRF .
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http://dx.doi.org/10.1038/s41598-020-75005-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578641PMC
October 2020

Polymerization retardation isothermal amplification strategy enables the sensitive and facile investigation of the flanking sequence preference of ten-eleven translocation 2 protein.

Anal Chim Acta 2020 May 21;1109:140-147. Epub 2020 Feb 21.

School of Chemistry, Sun Yat-Sen University, Guangzhou, 510275, China.

Active DNA demethylation process critically relies on the intrinsic properties of ten-eleven translocation proteins (Tets), particularly the flanking sequence preference. Challenged by the fact that the proximate bases to the 5-methylcytosine (5mC) are multitudinous and their influence on the Tets/DNA interplay is minute, the current methodologies are very limited in terms of cost, sensitivity and efficiency. Herein, we propose a polymerization retardation isothermal amplification (PRIA) strategy that enables sensitive and fast study of the flanking sequence preference of Tet. By arranging DNA polymerase to repetitively pass DNA strands through an isothermal replication-scission amplification reaction, the tiny difference in the Tet/DNA interplay can be consecutively accumulated and amplified. Low amount sample (80 ng) even multiple samples can be simultaneously analyzed within 10 h on an easily accessible laboratory real-time quantitative PCR instrument. For a proof-of-concept study, the binding preference (P) of Tet2 for XmCGX, (X = C, G, T, A) was analyzed by PRIA and computational analysis, showing an order of AmCGT > TmCGA ≈ GmCGC > CmCGG. Furthermore, the binding and oxidation preference in Tet/DNA interplay process was individually considered. By comparative evaluation of the total flanking sequence preference (P) and the P, for the first time, we revealed that Tet2 has a lower oxidation preference (P) to proximal flanking bases and the main contributor to P of Tet2 is P. The PRIA strategy, due to its reliable, cost-effective, high efficiency and low sample input, would hopefully advance epigenetic researches and other relative studies.
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http://dx.doi.org/10.1016/j.aca.2020.02.038DOI Listing
May 2020

Identification of Drug-Disease Associations Using Information of Molecular Structures and Clinical Symptoms via Deep Convolutional Neural Network.

Front Chem 2019 10;7:924. Epub 2020 Jan 10.

Key Laboratory of Digital Quality Evaluation of Chinese Materia Medica of State Administration of Traditional Chinese Medicine, Guangzhou, China.

Identifying drug-disease associations is helpful for not only predicting new drug indications and recognizing lead compounds, but also preventing, diagnosing, treating diseases. Traditional experimental methods are time consuming, laborious and expensive. Therefore, it is urgent to develop computational method for predicting potential drug-disease associations on a large scale. Herein, a novel method was proposed to identify drug-disease associations based on the deep learning technique. Molecular structure and clinical symptom information were used to characterize drugs and diseases. Then, a novel two-dimensional matrix was constructed and mapped to a gray-scale image for representing drug-disease association. Finally, deep convolution neural network was introduced to build model for identifying potential drug-disease associations. The performance of current method was evaluated based on the training set and test set, and accuracies of 89.90 and 86.51% were obtained. Prediction ability for recognizing new drug indications, lead compounds and true drug-disease associations was also investigated and verified by performing various experiments. Additionally, 3,620,516 potential drug-disease associations were identified and some of them were further validated through docking modeling. It is anticipated that the proposed method may be a powerful large scale virtual screening tool for drug research and development. The source code of MATLAB is freely available on request from the authors.
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http://dx.doi.org/10.3389/fchem.2019.00924DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6966717PMC
January 2020

Sensitive and sustained imaging of intracellular microRNA in living cells by a high biocompatible liposomal vehicle introduced isothermal symmetric exponential amplification reaction.

Chem Commun (Camb) 2019 Sep;55(75):11251-11254

School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, China.

A high biocompatible liposomal vehicle was used to deliver enzymes and oligonucleotide substances of the isothermal symmetric exponential amplification reaction into living cells, allowing sensitive and sustained imaging of microRNA in living cells for more than 24 h without apoptosis.
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http://dx.doi.org/10.1039/c9cc05583eDOI Listing
September 2019

Polymerization retardation isothermal amplification (PRIA): a strategy enables sensitively quantify genome-wide 5-methylcytosine oxides rapidly on handy instruments with nanoscale sample input.

Nucleic Acids Res 2019 11;47(19):e119

School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, China.

The current methods for quantifying genome-wide 5-methylcytosine (5mC) oxides are still scarce, mostly restricted with two limitations: assay sensitivity is seriously compromised with cost, assay time and sample input; epigenetic information is irreproducible during polymerase chain reaction (PCR) amplification without bisulfite pretreatment. Here, we propose a novel Polymerization Retardation Isothermal Amplification (PRIA) strategy to directly amplify the minute differences between epigenetic bases and others by arranging DNA polymerase to repetitively pass large electron-withdrawing groups tagged 5mC-oxides. We demonstrate that low abundant 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) in genomic DNA can be accurately quantified within 10 h with 100 ng sample input on a laboratory real-time quantitative PCR instrument, and even multiple samples can be analyzed simultaneously in microplates. The global levels of 5hmC and 5fC in mouse and human brain tissues, rat hippocampal neuronal tissue, mouse kidney tissue and mouse embryonic stem cells were quantified and the observations not only confirm the widespread presence of 5hmC and 5fC but also indicate their significant variation in different tissues and cells. The strategy is easily performed in almost all research and medical laboratories, and would provide the potential capability to other candidate modifications in nucleotides.
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http://dx.doi.org/10.1093/nar/gkz704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821303PMC
November 2019

Sandwich immunoassay coupled with isothermal exponential amplification reaction: An ultrasensitive approach for determination of tumor marker MUC1.

Talanta 2019 Nov 4;204:248-254. Epub 2019 Jun 4.

School of Chemistry and Chemical Engineering, Guangdong Pharmaceutical University, Guangzhou 510006, PR China. Electronic address:

An ultrasensitive strategy based on sandwich immunoassay coupled with isothermal exponential amplification reaction (IMEXPAR) is proposed for the determination of tumor protein Mucin 1 (MUC1). An immuno-PCR plate was prepared from modification of the primary MUC1-antibody (Ab) onto the inner-well of the PCR plate. A biotinylated secondary MUC1-antibody tagged with the biotinylated EXPAR primer (P-Ab) was prepared through biotin-streptavidin reaction. In the presence of target MUC1, sandwich-type combinations were specifically formed in the immuno-PCR plate. With further addition of amplification template, polymerase and nicking enzyme, EXPAR was specifically triggered, producing numerous primer replica in minutes, and greatly enhanced fluorescence of SYBR Green I. The proposed strategy has a good linear relationship with the logarithm of the MUC1 concentration ranging from 3 pM to 3 nM with a limit of detection of 1.63 pM (S/N = 3), which is two orders of magnitude lower than those of other methods. Owing to the specificity of immuno-reaction and EXPAR, the selectivity of the strategy is favorable, even if for the homologous protein. The proposed strategy was further applied for the MUC1 determination in human serum, and a satisfactory recovery range of 98.7%-105.3% was obtained. The strategy can be facilely extended to the ultrasensitive determination of various proteins.
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http://dx.doi.org/10.1016/j.talanta.2019.06.001DOI Listing
November 2019

Metastable Dumbbell Probe-Based Hybridization Chain Reaction for Sensitive and Accurate Imaging of Intracellular-Specific MicroRNAs In Situ in Living Cells.

Anal Chem 2019 04 20;91(7):4625-4631. Epub 2019 Mar 20.

School of Chemistry , Sun Yat-Sen University , Guangzhou 510275 , People's Republic of China.

Sensitive and accurate imaging of intracellular-specific microRNAs (miRNAs) in situ in living cells is seriously challenged by the susceptibility of nucleic acid probes and the low dynamics of the hybridization reaction in cellular environments. Herein, we engineer a set of new metastable dumbbell probes (M DPs) to overcome these key limitations by concurrently boosting transfection, antidigestibility, assembly dynamics, and nanostructural uniformity. The M DPs can maintain their stability up to 16 h in living cells and produce uniform and dense DNA nanostructures rapidly (<2 h) and specifically from a hybridization chain reaction (HCR). A sharp signal from the cascade accumulation of fluorescence resonance energy transfer (FRET) further minimizes the effect of system fluctuations. The M DPs-based HCR (M DPHCR) method showed identical performance in the analysis of miR-27a in cell lysate and buffer condition and obtained a limit of detection down to 3.2 pM (corresponding to 160 amol per 50 μL), which is 44-fold lower than on conventional hairpin probes. The M DPHCR method clearly distinguished normal cells from tumor cells and provided more accurate quantitative information on the intracellular-specific miRNAs. The strategy would offer a powerful tool for visualizing and localizing desired nucleic acids in living cells.
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http://dx.doi.org/10.1021/acs.analchem.8b05920DOI Listing
April 2019

In Situ Enzyme Immobilization with Oxygen-Sensitive Luminescent Metal-Organic Frameworks to Realize "All-in-One" Multifunctions.

Chemistry 2019 Apr 8;25(21):5463-5471. Epub 2019 Mar 8.

School of Chemistry, Sun Yat-sen University, Guangzhou, 510275, P. R. China.

Metal-organic frameworks (MOFs) for enzyme immobilization have already shown superior tunable and designable characteristics, however, their diverse responsive properties have rarely been exploited. In this work we integrated a responsive MOF into a MOF-enzyme composite with the purpose of designing an "all-in-one" multifunctional composite with catalytic and luminescence functions incorporated into a single particle. As a proof-of-concept, glucose oxidase (GOx) was encapsulated in situ within an oxygen (O )-sensitive, noble-metal-free, luminescent Cu triazolate framework (MAF-2), denoted as [email protected] Owing to the rigid scaffold of MAF-2 and confinement effect, the [email protected] composite showed significantly improved stability (shelf life of 60 days and heat resistance up to 80 °C) as well as good selectivity and recyclability. More importantly, owing to the O sensitivity of MAF-2, the [email protected] composite exhibited a rapid and reversible response towards dissolved O , thereby allowing direct and ratiometric sensing of glucose without the need for chromogenic substrates, cascade enzymatic reactions, or electrode systems. High sensitivity with a detection limit of 1.4 μm glucose was achieved, and the glucose levels in human sera were accurately determined. This strategy has led to a new application for MOFs that can be facilely extended to other MOF-enzyme composites due to the multifunctionality of MOFs.
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http://dx.doi.org/10.1002/chem.201806146DOI Listing
April 2019

Imaging of intracellular-specific microRNA in tumor cells by symmetric exponential amplification-assisted fluorescence in situ hybridization.

Chem Commun (Camb) 2018 Dec;54(99):13981-13984

School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, China.

We report a symmetric exponential amplification-assisted fluorescence in situ hybridization (SEXPAR-FISH) strategy for imaging intracellular-specific microRNAs (miRNAs) in cells. The strategy eliminates the risk of cell loss and miRNA leakage, and low-abundant intracellular miR-27a in cells can be imaged in only 2 h without compromising sensitivity and specificity.
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http://dx.doi.org/10.1039/c8cc08849gDOI Listing
December 2018

Real-Time Sensing of TET2-Mediated DNA Demethylation In Vitro by Metal-Organic Framework-Based Oxygen Sensor for Mechanism Analysis and Stem-Cell Behavior Prediction.

Anal Chem 2018 08 19;90(15):9330-9337. Epub 2018 Jul 19.

School of Chemistry , Sun Yat-sen University , Guangzhou 510275 , P. R. China.

Active DNA demethylation, mediated by O-dependent ten-eleven translocation (TET) enzymes, has essential roles in regulating gene expression. TET kinetics assay is vital for revealing mechanisms of demethylation process. Here, by a metal-organic framework (MOF)-based optical O sensor, we present the first demonstration on real-time TET2 kinetics assay in vitro. A series of luminescent Cu(I) dialkyl-1,2,4-triazolate MOFs were synthesized, which were noble-metal-free and able to intuitively response to dissolved O in a wide range from cellular hypoxia (≤15 μM) to ambient condition (∼257 μM). By further immobilization of the MOFs onto transparent silicon rubber ([email protected]) to construct O film sensors, and real-time monitoring of O consumption on [email protected] over the reaction time, the complete TET2-mediated 5-methylcytosine (5mC) oxidation process were achieved. The method overcomes the limitations of the current off-line methods by considerably shortening the analytical time from 0.5-18 h to 10 min, and remarkably reducing the relative standard deviation from 10%-68% to 0.68%-4.2%. As a result, the Michaelis-Menten constant ( K) values of TET2 for 5mC and O in ascorbic acid-free (AA) condition were precisely evaluated to be 24 ± 1 and 43.8 ± 0.3 μM, respectively. By comparative study on AA-containing (AA) conditions, and further establishing kinetics models, the stem-cell behavior of TETs was successfully predicted, and the effects of key factors (AA, O, Fe) on TETs were revealed, which were fully verified in mouse embryonic stem (mES) cells. The method is promising in wide application in kinetics analysis and cell behavior prediction of other important O-related enzymes.
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http://dx.doi.org/10.1021/acs.analchem.8b01941DOI Listing
August 2018

Short-probe-based duplex-specific nuclease signal amplification strategy enables imaging of endogenous microRNAs in living cells with ultrahigh specificity.

Talanta 2018 Aug 25;186:256-264. Epub 2018 Apr 25.

School of Chemistry, Sun Yat-Sen University, 135 Xingang West Road, Guangzhou 510275, PR China.

Specific nucleic acids amplification at a constant and mild temperature is important for imaging assay of endogenous microRNAs (miRNAs) in living cells. Duplex-specific nuclease (DSN) is attractive in one-step isothermal assay of miRNA; however, its intrinsic limitations of low amplification specificity and high reaction temperature greatly restrict the application scope. Herein, we present a short-probe-based DSN signal amplification (spDSNSA) strategy enabling analysis of miRNAs at body temperature with significantly high specificity. From systematic investigation of amplification reaction on different types of DNA probes, we revealed that the annealing rate between probe and target miRNA greatly affects the dynamics of amplification process. By simply shortening the length of DNA probe, the spDSNSA remarkably improved specificity without loss of amplification efficiency at 37 °C. As a proof-of-concept, let-7a was sensitively detected by spDSNSA with a limit of detection down to 30 p.M., and a specificity 10 ‒ 10 folds higher than those of traditional DSNSA methods. The analysis of the let-7a in the lysates of A549 human lung cancer cells and BEAS-2B human lung normal bronchial epithelial cells exhibited well agreement with rt-qPCR method. Furthermore, the endogenous let-7a in A549 and BEAS-2B living cells was clearly imaged without damaging the original morphology of cells. The method provide a facile idea for extension of DNS related signal amplification strategies in the application in living cells and POCTs, and would pose a great impact on the development of simple and rapid molecular diagnostic applications for short oligonucleotides.
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http://dx.doi.org/10.1016/j.talanta.2018.04.071DOI Listing
August 2018

Isothermal Amplification on a Structure-Switchable Symmetric Toehold Dumbbell-Template: A Strategy Enabling MicroRNA Analysis at the Single-Cell Level with Ultrahigh Specificity and Accuracy.

Anal Chem 2018 01 13;90(1):859-865. Epub 2017 Dec 13.

School of Chemistry, Sun Yat-Sen University , Guangzhou 510275, PR China.

Accurate analysis of microRNAs (miRNAs) at the single-cell level seriously requires analytical methods possessing extremely high sensitivity, specificity and precision. By rational engineering of a structure-switchable symmetric toehold dumbbell-template (STD-template), we propose a novel isothermal symmetric exponential amplification reaction (SEXPAR) method. The sealed and symmetric structure of the STD-template allows exponential amplification reaction (EXPAR) to occur upon every annealing of target miRNA without loss of amplification efficiency. In addition, the rigid and compact structure of the STD-template with an appropriate standard free energy ensures SEXPAR only be activated by target miRNA. As a result, the SEXPAR method isothermally quantified let-7a down to 0.01 zmol (6.02 copies per 10 μL) with an ultrahigh specificity which is efficient enough to discriminate one-base-mismatched miRNAs, and a remarkably high precision even for the determination of 6.02 copies let-7a (the standard deviation was reduced from >60% down to 23%). The dynamic range was also extended to 10 orders of magnitude. The method was successfully applied for the determination of let-7a in human tissues, sera and even single-cell lysate, with obviously better precision than quantitative reverse transcription polymerase chain reaction (RT-qPCR) and other EXPAR-based methods. The SEXPAR method may serve as a powerful technique for the biological research and biomedical studies of miRNAs and other short nucleic acids.
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http://dx.doi.org/10.1021/acs.analchem.7b03713DOI Listing
January 2018

Electrochemical assay for continuous monitoring of dynamic DNA methylation process.

Biosens Bioelectron 2018 Feb 4;100:184-191. Epub 2017 Sep 4.

School of Chemistry and Chemical Engineering, Sun Yat-Sen University, 135 Xingang West Road, Guangzhou 510275, PR China. Electronic address:

A simple electrochemical strategy is reported for continuous monitoring of dynamic DNA methylation process over time. An electrochemical sensor was prepared by co-assembling of DNA probe and 6-mercapto-1-hexanol onto a gold electrode. The top of the DNA probe was labeled with 6-ferrocenylhexanethiol modified gold nanoparticle. The charge density between the C•G base pair was verified to be slightly reduced by DNA methylation, and could be further decelerated by ~ 25% upon co-locating a Br group onto methylated cytosine (mC). Therefore, in the presence of NaIO/LiBr, the progressively methylated DNA on the sensor showed a clearly decreasing current over methylation time. The dynamic DNA methylation process was indicated continuously from the current decrease ratio, with a limit of detection of 0.0372µM. The strategy is convenient, cost-effective, and enable continuous profiling methylation process without distortion. Besides, the strategy was successfully applied for the studies on inhibitor screening and flanking sequence preference of DNA methyltransferase 3a. The results show that the activity of DNA methyltransferase 3a can be mildly inhibited by epigallocatechin gallate, and varies towards different flanking sequence with an order of 5'-CCGG-3' < 5'-CGCG-3' < 5'-CGCA-3'.
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http://dx.doi.org/10.1016/j.bios.2017.08.013DOI Listing
February 2018

Large-scale identification of human protein function using topological features of interaction network.

Sci Rep 2016 11 16;6:37179. Epub 2016 Nov 16.

SYSU-CMU Shunde International Joint Research Institute, Shunde, 528300, People's Republic of China.

The annotation of protein function is a vital step to elucidate the essence of life at a molecular level, and it is also meritorious in biomedical and pharmaceutical industry. Developments of sequencing technology result in constant expansion of the gap between the number of the known sequences and their functions. Therefore, it is indispensable to develop a computational method for the annotation of protein function. Herein, a novel method is proposed to identify protein function based on the weighted human protein-protein interaction network and graph theory. The network topology features with local and global information are presented to characterise proteins. The minimum redundancy maximum relevance algorithm is used to select 227 optimized feature subsets and support vector machine technique is utilized to build the prediction models. The performance of current method is assessed through 10-fold cross-validation test, and the range of accuracies is from 67.63% to 100%. Comparing with other annotation methods, the proposed way possesses a 50% improvement in the predictive accuracy. Generally, such network topology features provide insights into the relationship between protein functions and network architectures. The source code of Matlab is freely available on request from the authors.
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http://dx.doi.org/10.1038/srep37179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5111120PMC
November 2016

Asymmetric exponential amplification reaction on a toehold/biotin featured template: an ultrasensitive and specific strategy for isothermal microRNAs analysis.

Nucleic Acids Res 2016 09 2;44(15):e130. Epub 2016 Jun 2.

School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, China SYSU-CMU Shunde International Joint Research Institute, Shunde, Guangdong 528300, China.

The sensitive and specific analysis of microRNAs (miRNAs) without using a thermal cycler instrument is significant and would greatly facilitate biological research and disease diagnostics. Although exponential amplification reaction (EXPAR) is the most attractive strategy for the isothermal analysis of miRNAs, its intrinsic limitations of detection efficiency and inevitable non-specific amplification critically restrict its use in analytical sensitivity and specificity. Here, we present a novel asymmetric EXPAR based on a new biotin/toehold featured template. A biotin tag was used to reduce the melting temperature of the primer/template duplex at the 5' terminus of the template, and a toehold exchange structure acted as a filter to suppress the non-specific trigger of EXPAR. The asymmetric EXPAR exhibited great improvements in amplification efficiency and specificity as well as a dramatic extension of dynamic range. The limit of detection for the let-7a analysis was decreased to 6.02 copies (0.01 zmol), and the dynamic range was extended to 10 orders of magnitude. The strategy enabled the sensitive and accurate analysis of let-7a miRNA in human cancer tissues with clearly better precision than both standard EXPAR and RT-qPCR. Asymmetric EXPAR is expected to have an important impact on the development of simple and rapid molecular diagnostic applications for short oligonucleotides.
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http://dx.doi.org/10.1093/nar/gkw504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5009742PMC
September 2016

Triple cascade reactions: An ultrasensitive and specific single tube strategy enabling isothermal analysis of microRNA at sub-attomole level.

Biosens Bioelectron 2016 Jun 24;80:378-384. Epub 2016 Jan 24.

School of Chemistry and Chemical Engineering, Sun Yat-Sen University, 135 Xingang West Road, Guangzhou 510275, PR China; SYSU-CMU Shunde International Joint Research Institute, Shunde, Guangdong 528300, PR China. Electronic address:

Sensitive and specific analysis of microRNAs (miRNAs) in a single tube without the need of thermal cycler instrument would greatly facilitate the investigation of miRNA-associated regulatory circuits and diseases. Homogeneous isothermal amplification assays are attractive in conducting single tube assays that can minimize contamination-prone steps and simplifies assay procedures. However, the relative low amplification efficiency and high detection background remain as bottlenecks restricting their more versatile applications. In this work, we have developed a novel isothermal exponential enzymatic amplification (IEEA) strategy for miRNAs analysis. By rational triple cascade amplification cycles of target recycling, nicking-replication reaction, and DNAzyme catalysis, the strategy exhibited high signal amplification efficiency (10(4)-10(9) folds of amplification in 1h) with very low detection background and excellent specificity. As a result, the miR-27a target model was rapidly determined with a limit of detection down to 0.79 aM (S/N=3), corresponding to 94 copies of the miRNA molecule in a 200 μL sample solution. The levels of miR-27a in atherosclerotic sprague-dawley rats were accurately quantified. The strategy is anticipated to have an important impact on the development of simple and rapid molecular diagnostic applications for any short oligonucleotides.
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http://dx.doi.org/10.1016/j.bios.2016.01.059DOI Listing
June 2016

Chemical-oxidation cleavage triggered isothermal exponential amplification reaction for attomole gene-specific methylation analysis.

Anal Chem 2015 Mar 11;87(5):2945-51. Epub 2015 Feb 11.

School of Chemistry and Chemical Engineering, Sun Yat-Sen University , Guangzhou, 510275 Guangdong, PR China.

Genomic 5-methylcytosine (5-mC) modification is known to extensively regulate gene expression. The sensitive and convenient analysis of gene-specific methylation is wishful but challenging due to the lack of means that can sensitively and sequence-selectively discriminate 5-mC from cytosine without the need for polymerase chain reaction. Here we report a chemical-oxidation cleavage triggered exponential amplification reaction (EXPAR) method named COEXPAR for gene-specific methylation analysis. EXPAR was proved to not only have rapid amplification kinetics under isothermal condition but also show excellent sequence-selectivity and linear-dependence on EXPAR trigger. Further initiation of EXPAR by chemical-cleavage of DNA at 5-mC, the COEXPAR showed high specificity for methylated and nonmethylated DNA, and ∼10(7) copies of triggers were replicated in 20 min, which were used to quantify the methylation level at the methylation loci. As a result, the gene-specific methylation level of a p53 gene fragment, as a target model, was analyzed in two linear ranges of 10 fM-1 pM and 1 pM-10 nM, and limits of detection of 411 aM (S/N = 3) by fluorescence, and 576 aM (S/N = 3) by electrochemistry. The method fulfilled the assay in an isothermal way in ∼5 h without the need for tedious sample preparation and accurate thermocycling equipment, which is likely to be a facile and ultrasensitive way for gene-specific methylation analysis.
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http://dx.doi.org/10.1021/ac5044785DOI Listing
March 2015

Construction of a zinc porphyrin-fullerene-derivative based nonenzymatic electrochemical sensor for sensitive sensing of hydrogen peroxide and nitrite.

Anal Chem 2014 Jul 19;86(13):6285-90. Epub 2014 Jun 19.

College of Chemistry & Chemical Engineering, Fuyang Normal College , Fuyang, Anhui 236037, P.R. China.

Enzymatic sensors possess high selectivity but suffer from some limitations such as instability, complicated modified procedure, and critical environmental factors, which stimulate the development of more sensitive and stable nonenzymatic electrochemical sensors. Herein, a novel nonenzymatic electrochemical sensor is proposed based on a new zinc porphyrin-fullerene (C60) derivative (ZnP-C60), which was designed and synthesized according to the conformational calculations and the electronic structures of two typical ZnP-C60 derivatives of para-ZnP-C60 (ZnP(p)-C60) and ortho-ZnP-C60 (ZnP(o)-C60). The two derivatives were first investigated by density functional theory (DFT) and ZnP(p)-C60 with a bent conformation was verified to possess a smaller energy gap and better electron-transport ability. Then ZnP(p)-C60 was entrapped in tetraoctylammonium bromide (TOAB) film and modified on glassy carbon electrode (TOAB/ZnP(p)-C60/GCE). The TOAB/ZnP(p)-C60/GCE showed four well-defined quasi-reversible redox couples with extremely fast direct electron transfer and excellent nonenzymatic sensing ability. The electrocatalytic reduction of H2O2 showed a wide linear range from 0.035 to 3.40 mM, with a high sensitivity of 215.6 μA mM(-1) and a limit of detection (LOD) as low as 0.81 μM. The electrocatalytic oxidation of nitrite showed a linear range from 2.0 μM to 0.164 mM, with a sensitivity of 249.9 μA mM(-1) and a LOD down to 1.44 μM. Moreover, the TOAB/ZnP(p)-C60/GCE showed excellent stability and reproducibility, and good testing recoveries for analysis of the nitrite levels of river water and rainwater. The ZnP(p)-C60 can be used as a novel material for the fabrication of nonenzymatic electrochemical sensors.
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http://dx.doi.org/10.1021/ac500245kDOI Listing
July 2014

A label-free and PCR-free electrochemical assay for multiplexed microRNA profiles by ligase chain reaction coupling with quantum dots barcodes.

Biosens Bioelectron 2014 Mar 24;53:414-9. Epub 2013 Oct 24.

School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, PR China; College of Chemistry and Bioengineering, Guilin University of Technology, Guilin 541004, PR China.

The profiling of microRNAs (miRNAs) is greatly significant for cellular events or disease diagnosis. Electrochemical methods for miRNAs analysis mostly can only measure one kind of miRNA, which is unambiguous to indicate the disease type and state. Here a label-free and PCR-free electrochemical method is presented for multiplexed evaluation of miRNAs in a single-tube experiment. The method is based on the combination of the high base-mismatch selectivity of ligase chain reaction (LCR) and the remarkable voltammetric signature of electrochemical QDs barcodes. Two reporting probes of RP1 and RP2 were labeled with PbS and CdS quantum dots (QDs) to prepare PbS-RP1 and CdS-RP2 conjugates, and two capture probes of CP1 and CP2 were co-immobilized on magnetic beads (MBs) to fabricate MB-CP1CP2 conjugate. The miRNAs samples were simply incubated with MB-CP1CP2, PbS-RP1, and CdS-RP2 conjugates, and then added with T4 DNA ligase. After release of the disjoined QDs barcodes from the MB-conjugates, two target miRNAs of miR-155 and miR-27b were simultaneously detected by square wave voltammetry with linear ranges of 50 fM-30 pM and 50 fM-1050 pM, and limits of detection (LODs) of 12 fM and 31 fM (S/N=3). The method fulfilled the assay in less than 70 min, and showed acceptable testing recoveries for the determination of miRNAs in biological matrix. Currently there are rare reports about electrochemical multiplexed quantification of miRNAs. The method is likely to provide a new platform for identification of multiple miRNAs in a simple way.
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http://dx.doi.org/10.1016/j.bios.2013.10.023DOI Listing
March 2014
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