Duanping Sun

Ultrasensitive electrochemical detection of microRNA based on an arched probe mediated isothermal exponential amplification.

In this work, a simple and label-free electrochemical biosensor is developed for microRNA (miRNA) detection on the basis of an arched probe mediated isothermal exponential amplification reaction (EXPAR). The arched probe assembled on the electrode surface consists of two strands that are partially complementary to each other at both ends. The target can hybridize with the complementary sequence of the arched structure, leading to the cleavage of the probe. The strand fixed on the surface of the electrode self-assembles, in the presence of hemin, to G-quadruplex unit, yielding electrochemical signals. The other strand liberated into the solution triggers the EXPAR to recycle and regenerate targets. This method exhibits ultrahigh sensitivity toward miRNA with detection limits of 5.36 fM and a detection range of 3 orders of magnitude. The biosensor is capable of discriminating a single-nucleotide difference between concomitant miRNA and performs well in analyzing crude extractions from cancer cell lines.

Sensitive electrochemical aptamer cytosensor for highly specific detection of cancer cells based on the hybrid nanoelectrocatalysts and enzyme for signal amplification

Human cancer is becoming a leading cause of death in the world and the development of a straightforward strategy for early detection of cancer is urgently required. Herein, a sandwich-type electrochemical aptamer cytosensor was developed for detection of human liver hepatocellular carcinoma cells (HepG2) based on the hybrid nanoelectrocatalysts and enzyme for signal amplification. The thiolated TLS11a aptamers were used as a selective bio-recognition element, attached to the gold nanoparticles (AuNPs) modified the glassy carbon electrode (GCE) surface. Meanwhile, the electrochemical nanoprobes were fabricated through the G-quadruplex/hemin/aptamer complexes and horseradish peroxidase (HRP) immobilized on the surfaces of Au@Pd core-shell nanoparticle-modified magnetic Fe3O4/MnO2 beads (Fe3O4/MnO2/Au@Pd). After the target cells were captured, the hybrid nanoprobes were further assembled to form an aptamer-cell-nanoprobes sandwich-like system on the electrode surface. Then, hybrid Fe3O4/MnO2/Au@Pd nanoelectrocatalysts, G-quadruplex/hemin HRP-mimicking DNAzymes and the natural HRP enzyme efficiently catalyzed the oxidation of hydroquinone (HQ) with H2O2, amplifying the electrochemical signals and improving the detection sensitivity. This electrochemical cytosensor delivered a wide detection range of 1×10(2)-1×10(7)cellsmL(-1), high sensitivity with a low detection limit of 15cellsmL(-1), good selectivity and repeatability. Finally, an electrochemical reductive desorption method was performed to break gold-thiol bond and desorb the components on the AuNPs/GCE for regenerating the cytosensor. These results have demonstrated that the electrochemical cytosensor has the potential to be a feasible tool for cost-effective cancer cell detection in early cancer diagnosis.

A repeatable assembling and disassembling electrochemical aptamer cytosensor for ultrasensitive and highly selective detection of human liver cancer cells.

In this work, a repeatable assembling and disassembling electrochemical aptamer cytosensor was proposed for the sensitive detection of human liver hepatocellular carcinoma cells (HepG2) based on a dual recognition and signal amplification strategy. A high-affinity thiolated TLS11a aptamer, covalently attached to a gold electrode through Au-thiol interactions, was adopted to recognize and capture the target HepG2 cells. Meanwhile, the G-quadruplex/hemin/aptamer and horseradish peroxidase (HRP) modified gold nanoparticles (G-quadruplex/hemin/aptamer-AuNPs-HRP) nanoprobe was designed. It could be used for electrochemical cytosensing with specific recognition and enzymatic signal amplification of HRP and G-quadruplex/hemin HRP-mimicking DNAzyme. With the nanoprobes as recognizing probes, the HepG2 cancer cells were captured to fabricate an aptamer-cell-nanoprobes sandwich-like superstructure on a gold electrode surface. The proposed electrochemical cytosensor delivered a wide detection range from 1×10(2) to 1×10(7) cells mL(-1) and high sensitivity with a low detection limit of 30 cells mL(-1). Furthermore, after the electrochemical detection, the activation potential of -0.9 to -1.7V was performed to break Au-thiol bond and regenerate a bare gold electrode surface, while maintaining the good characteristic of being used repeatedly. The changes of gold electrode behavior after assembling and desorption processes were investigated by electrochemical impedance spectroscopy and cyclic voltammetry techniques. These results indicate that the cytosensor has great potential in disease diagnostic of cancers and opens new insight into the reusable gold electrode with repeatable assembling and disassembling in the electrochemical sensing.

Zebrafish on a Chip: A Novel Platform for Real-Time Monitoring of Drug-Induced Developmental Toxicity

Pharmaceutical safety testing requires a cheap, fast and highly efficient platform for real-time evaluation of drug toxicity and secondary effects. In this study, we have developed a microfluidic system for phenotype-based evaluation of toxic and teratogenic effects of drugs using zebrafish (Danio rerio) embryos and larvae as the model organism. The microfluidic chip is composed of two independent functional units, enabling the assessment of zebrafish embryos and larvae. Each unit consists of a fluidic concentration gradient generator and a row of seven culture chambers to accommodate zebrafish. To test the accuracy of this new chip platform, we examined the toxicity and teratogenicity of an anti-asthmatic agent-aminophylline (Apl) on 210 embryos and 210 larvae (10 individuals per chamber). The effect of Apl on zebrafish embryonic development was quantitatively assessed by recording a series of physiological indicators such as heart rate, survival rate, body length and hatch rate. Most importantly, a new index called clonic convulsion rate, combined with mortality was used to evaluate the toxicities of Apl on zebrafish larvae. We found that Apl can induce deformity and cardiovascular toxicity in both zebrafish embryos and larvae. This microdevice is a multiplexed testing apparatus that allows for the examination of indexes beyond toxicity and teratogenicity at the sub-organ and cellular levels and provides a potentially cost-effective and rapid pharmaceutical safety assessment tool.

Microfluidic contactless conductivity cytometer for electrical cell sensing and counting

The microfluidic cytometer has recently attracted increasing attention in cell sensing and counting due to its advantages of high-speed measurement, small sampling size and being cost effective. Up to now, the conventional microfluidic cytometer has usually used expensive microelectrodes directly in contact with a cell suspension to measure the changes in electrical signals. In this paper, we introduce a novel approach to construct an integrated microfluidic contactless conductivity cytometer for non-invasive analysis of samples with a small number of cells. The microfluidic chip is composed of a polydimethylsiloxane (PDMS) plate with a narrow microchannel on the top, a 100 μm thick glass plate in the middle and a glass substrate containing indium tin oxide (ITO) detecting electrodes on the bottom. This contactless measurement approach avoids direct contact between the detection buffer and the ITO electrodes, allowing the electrodes to be easily reused and lowering the cost of the device. When cells flow through a narrow microfluidic channel which is a little larger than the tested cell size, electrical signals are detected by a capacitively coupled contactless conductivity detection (C4D) system. Furthermore, human breast cancer (MCF-7) cells and hypertrophic cardiomyocytes (HCM) were used to successfully demonstrate the feasibility of the microfluidic contactless conductivity sensor for counting and detecting cells. Consequently, the designed microfluidic cytometer is cost-effective, easy-to-use and label-free. It is reasonable to expect that this microfluidic cytometer can become a promising tool for label-free cell counting and point-of-care clinical diagnosis in the developing world.

A two-electrode system-based electrochemiluminescence detection for microfluidic capillary electrophoresis and its application in pharmaceutical analysis.

A two-electrode configuration powered by batteries was designed for a microchip capillary electrophoresis-electrochemiluminescence system. A home-made working electrode for end-column mode detection and wall-jet configuration was made up of a platinum wire (0.3 mm diameter) and a quartz capillary (320 µm internal diameter). The platinum wire served as a pseudoreference electrode. The configuration of the detection power supply comprised two D-size batteries (connected in series), a switch, and an adjustable resistor. The microchip consisted of two layers: the bottom layer was a glass sheet containing injection and separation channels; the upper layer was polydimethylsiloxane block. In order to reduce the loss of electrochemiluminescence signal, a coverslip (0.17 mm thickness) was used as the floor of the detection reservoir. The performance of the system was demonstrated by separation and detection of atropine, anisodamine and proline. The linear response for proline ranged from 5 µM to 100 µM (r = 0.9968), and the limit of detection was 1.0 µM (S/N = 3). The system was further applied to the measurement of atropine in atropine sulfate injection solutions with the limit of detection 2.3 µM. This new system is a potential tool in pharmaceutical analysis.

Nanomaterial-based Microfluidic Chips for the Capture and Detection of Circulating Tumor Cells

Circulating tumor cells (CTCs), a type of cancer cells that spreads from primary or metastatic tumors into the bloodstream, can lead to a new fatal metastasis. As a new type of liquid biopsy, CTCs have become a hot pursuit and detection of CTCs offers the possibility for early diagnosis of cancers, earlier evaluation of chemotherapeutic efficacy and cancer recurrence, and choice of individual sensitive anti-cancer drugs. The fundamental challenges of capturing and characterizing CTCs are the extremely low number of CTCs in the blood and the intrinsic heterogeneity of CTCs. A series of microfluidic devices have been proposed for the analysis of CTCs with automation capability, precise flow behaviors, and significant advantages over the conventional larger scale systems. This review aims to provide in-depth insights into CTCs analysis, including various nanomaterial-based microfluidic chips for the capture and detection of CTCs based on the specific biochemical and physical properties of CTCs. The current developmental trends and promising research directions in the establishment of microfluidic chips for the capture and detection of CTCs are also discussed.

Nonenzymatic Electrochemical Immunosensor Using Ferroferric Oxide–Manganese Dioxide–Reduced Graphene Oxide Nanocomposite as Label for α-Fetoprotein Detection

A novel nonenzymatic electrochemical immunosensor was fabricated for quantitative detection of α-fetoprotein (AFP). The immunosensor was constructed by modifying gold electrode with electrochemical reduction of graphene oxide-carboxyl multi-walled carbon nanotube composites (ERGO–CMWCNTs) and electrodeposition of gold nanoparticles (AuNPs) for effective immobilization of primary antibody (Ab1). Ferroferric oxide–manganese dioxide–reduced graphene oxide nanocomposites (Fe3O4@MnO2–rGO) were designed as labels for signal amplification. On one hand, the excellent electroconductivity and outstanding electron transfer capability of ERGO–CMWCNTs/AuNPs improved the sensitivity of the immunosensor. On the other hand, introduction of rGO could not only increase the specific surface area for immobilization of secondary antibody (Ab2) but also build a synergetic effect to reinforce the electrocatalytic properties of catalysts. Fe3O4@MnO2–rGO nanocomposites were characterized by scanning electron microscope, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Using AFP as a model analyte, the proposed sandwich-type electrochemical immunosensor exhibited a wide linear range of 0.01–50ng⋅mL−1 with a low detection limit of 5.8pg⋅mL−1. Moreover, the Fe3O4@MnO2–rGO-based peroxidase mimetic system displayed an excellent analytical performance with low cost, satisfactory reproducibility and high selectivity, which could be further extended for detecting other disease-related biomarkers.

Comparative toxicity of lead (Pb2+), copper (Cu2+), and mixtures of lead and copper to zebrafish embryos on a microfluidic chip

Investigations were conducted to determine acute effects of Pb(2+) and Cu(2+) presented individually and collectively on zebrafish embryos. Aquatic safety testing requires a cheap, fast, and highly efficient platform for real-time evaluation of single and mixture of metal toxicity. In this study, we have developed a microfluidic system for phenotype-based evaluation of toxic effects of Pb(2+) and Cu(2+) using zebrafish (Danio rerio) embryos. The microfluidic chip is composed of a disc-shaped concentration gradient generator and 24 culture chambers, which can generate one blank solution, seven mixture concentrations, and eight single concentrations for each metal solution, thus enabling the assessment of zebrafish embryos. To test the accuracy of this new chip platform, we have examined the toxicity and teratogenicity of Pb(2+) and Cu(2+) on embryos. The individual and combined impact of Pb(2+) and Cu(2+) on zebrafish embryonic development was quantitatively assessed by recording a series of physiological indicators, such as spontaneous motion at 22 hours post fertilization (hpf), mortality at 24 hpf, heartbeat and body length at 96 hpf, etc. It was found that Pb(2+) or Cu(2+) could induce deformity and cardiovascular toxicity in zebrafish embryos and the mixture could induce more severe toxicity. This chip is a multiplexed testing apparatus that allows for the examination of toxicity and teratogenicity for substances and it also can be used as a potentially cost-effective and rapid aquatic safety assessment tool.

A DNA nanostructured aptasensor for the sensitive electrochemical detection of HepG2 cells based on multibranched hybridization chain reaction amplification strategy

Sensitive detection of cancer cells is beneficial to the early diagnosis of cancer and individual treatment. In the present study, a DNA nanostructured aptasensor was used for the sensitive electrochemical detection of human liver hepatocellular carcinoma cells (HepG2) based on multibranched hybridization chain reaction amplification strategy. We established a well-designed platform by immobilizing DNA tetrahedron, a three-dimensional DNA nanostructure, on the gold electrode to capture HepG2 cells more specifically and efficiently. Meanwhile, functional hybrid nanoprobes consisted of MIL-101@AuNPs (Au nanoparticles), numerous hemin/G-quadruplex DNAzyme from multibranched hybridization chain reaction, and natural horseradish peroxidase (HRP) was designed. The hybrid nanoprobes possessed the functions of specific discernment and enzymatic signal amplification simultaneously. With the help of nanoprobes, HepG2 cells were recognized and captured to form a DNA tetrahedron-cell-nanoprobe sandwich-like structure on the electrode surface. The lower detection limit of this established cytosensor is 5 cells per ml. Moreover, it delivered a broad detection range from 102 to 107 cells per ml. The results revealed that the as-proposed cytosensor may be utilized as a powerful tool for early diagnosis of cancer in the future.