Yuhui Liao

Target-triggered enzyme-free amplification strategy for sensitive detection of microRNA in tumor cells and tissues.

MicroRNAs (miRNAs) participate in important processes of life course. Because of their characters of small sizes, vulnerable degradabilities, and sequences similarities, the existing detection technologies mostly contain enzymatic amplification reactions for acquisition of high sensitivities and specificities. However, specific reaction conditions and time-dependent enzyme activities are caused by the accession of enzymes. Herein, we designed a target-triggered enzyme-free amplification platform that is realized by circulatory interactions of two hairpin probes and the integrated electrochemiluminescence (ECL) signal giving-out component. Benefiting from outstanding performances of the enzyme-free amplification system and ECL, this strategy is provided with a simplified reaction process, high sensitivity, and operation under isothermal conditions. Through detection of the miRNA standard substance, the sensitivity of this platform reached 10 fmol, and a splendid specificity was achieved. We also analyzed three tumor cell lines (human lung adenocarcinoma, breast adenocarcinoma, and hepatocellular liver carcinoma cell lines) through this platform. The sensitivities of 10(3) cells, 10(4) cells, and 10(4) cells were, respectively, achieved. Furthermore, clinical tumor samples were tested, and 21 of 30 experimental samples gave out positive signals. Thus, this platform possesses potentials to be an innovation in miRNA detection methodology.

Synthesis, labeling and bioanalytical applications of a tris(2,2′-bipyridyl)ruthenium(II)-based electrochemiluminescence probe

Assays using probes labeled with electrochemiluminescent moieties are extremely powerful analytical tools that are used in fields such as medical diagnostics, environmental analysis and food safety monitoring, in which sensitive, reliable and reproducible detection of biomolecules is a requirement. The most efficient electrochemiluminescence (ECL) reaction to date is based on tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)32+) with tripropylamine (TPrA) as the co-reactant. Here we present a detailed protocol for preparing Ru(bpy)32+ probes and their bioanalytical applications. This protocol includes (i) the synthesis of a biologically active Ru(bpy)32+-N-hydroxysuccinimide (NHS) ester, (ii) its covalent labeling with both antibodies and DNA probes and (iii) the detection and quantification of ECL in a microfluidic system with a paramagnetic microbead solid support. In our magnetic bead–based ECL system, two probes are required: a capture probe (labeled with biotin to be captured by a streptavidin-coated magnetic bead) and a detector probe (labeled with Ru(bpy)32+). The complex consisting of the analyte, the capture probe, the detector probe and the magnetic bead is brought into contact with the electrode by using a magnetic field. The Ru(bpy)32+ reacts with TPrA in solution to generate the ECL signal. The full protocol, including the synthesis and labeling of the bioactive Ru(bpy)32+, requires 5–6 d to complete. ECL immunoassays or nucleic acid tests only require 1.5–2 h, including the sample preparation time.

Quantum Dots and Graphene Oxide Fluorescent Switch Based Multivariate Testing Strategy for Reliable Detection of Listeria monocytogenes

Graphene oxide (GO) and quantum dots (QDs), as burgeoning types of nanomaterials, have gained tremendous interest in the biosensor field. In this work, we designed a novel multivariate testing strategy that depends on the fluorescence resonance energy transfer (FRET) effect between quantum dots (QDs) and graphene oxide (GO). It integrates the QD-GO FRET principle and QD probes with different emission peaks into a platform, aims at multiplex gene detection of a human infectious and highly pathogenic pathogen, Listeria monocytogenes (L. monocytogenes). With the development of a multiplex linear-after-the-exponential (LATE) polymerase chain reaction (PCR) system, the single-stranded DNA (ssDNA) products of hlyA genes and iap genes are obtained by simultaneous amplification of the target genes. Then with the hybridization of ssDNA products and QD probes, simultaneous homogeneous detection of two gene amplification products can be achieved by using GO as a fluorescence switch and monitoring the relevant emissions excited by a single light source. Distinguishable signals corresponding to target genes are obtained. With this developed approach, genomic DNA from L. monocytogenes can be detected as low as 100 fg/μL. Moreover, this platform has a good dynamic range from 10(2) to 10(6) fg/μL. It is indicated that this platform has potential to be a reliable complement for rapid gene detection technologies and is capable of reducing the false-negative and false-dismissal probabilities in routine tests.

Toehold-mediated nonenzymatic amplification circuit on graphene oxide fluorescence switching platform for sensitive and homogeneous microRNA detection.

A novel graphene oxide (GO) fluorescence switch-based homogenous system has been developed to solve two problems that are commonly encountered in conventional GO-based biosensors. First, with the assistance of toehold-mediated nonenzymatic amplification (TMNA), the sensitivity of this system greatly surpasses that of previously described GO-based biosensors, which are always limited to the nM range due to the lack of efficient signal amplification. Second, without enzymatic participation in amplification, the unreliability of detection resulting from nonspecific desorption of DNA probes on the GO surface by enzymatic protein can be avoided. Moreover, the interaction mechanism of the double-stranded TMNA products contains several single-stranded toeholds at two ends and GO has also been explored with combinations of atomic force microscopy imaging, zeta potential detection, and fluorescence assays. It has been shown that the hybrids can be anchored to the surface of GO through the end with more unpaired bases, and that the other end, which has weaker interaction with GO, can escape GO adsorption due to the robustness of the central dsDNA structures. We verified this GO fluorescence switch-based detection system by detecting microRNA 21, an overexpressed non-encoding gene in a variety of malignant cells. Rational design of the probes allowed the isothermal nonenzymatic reaction to achieve more than 100-fold amplification efficiency. The detection results showed that our strategy has a detection limit of 10 pM and a detection range of four orders of magnitude.

Ultrasensitive Detection of MicroRNA in Tumor Cells and Tissues via Continuous Assembly of DNA Probe

Nucleic acids have been engineered to participate in a wide variety of tasks. Among them, the enzyme-free amplification modes, enzyme-free DNA circuits (EFDCs), and hybridization chain reactions (HCRs) have been widely applied in a series of studies of bioanalysis. We demonstrated here an ultrasensitive hairpin probe-based circulation for continuous assemble of DNA probe. This strategy improved the analyte stability-dependent amplification efficiency of EFDC and signal enhancement without being limited by the analytes initial concentration, and it was used to produce a novel microRNA (miRNA) trace analysis assay with ultrasensitive amplification properties. Through the detection of standard miRNA substances, 1 amol-level sensitivity and satisfactory specificity were achieved. Compared with EFDCs and HCRs, the sensitivity of ultrasensitive hairpin probe-based circulation was higher by 3 or 4 orders of magnitude. Furthermore, the excellent performance of this platform was also demonstrated in the detection of miRNAs in tumor cells. The sensitivities for the detection of miRNAs in HepG2, A549 and MCF-7 tumor cells were 10, 10, and 100 cells, respectively. In addition, a high detection rate of 83% was achieved for tumor tissues. Thus, this ultrasensitive hairpin probe-based circulation possesses the potential to be a technological innovation in the field of tumor diagnosis.

Linear Ru(bpy)32+–Polymer as a Universal Probe for Sensitive Detection of Biomarkers with Controllable Electrochemiluminescence Signal-Amplifying Ratio

Electrochemiluminescence (ECL) has been engineered to perform various tasks in the area of immunoassays and molecular diagnosis. However, there is still substantial potential for developments of ECL assay with high efficiency to achieve trace analysis. Herein, we demonstrate a polymer-amplified ECL assay via construction of linear Ru(bpy)32+-polymer. This new polymer material compensates for the relatively low ECL intensity from single ECL luminophore and realizes a stable and controllable labeling process. The polymer-amplified ECL assay achieved a remarkable sensitivity of 100 amol. The wide-ranging applications of the polymer-amplified ECL assay for Hepatitis B virus, carcinoembryonic antigen, 16sRNA, and thrombin also demonstrate its superiority. Hence, the polymer-amplified ECL assay possesses the potential to create a new paradigm in amplified ECL assays that could provide outstanding performance for biomedical analysis.

Graphene Oxide as a Bifunctional Material toward Superior RNA Protection and Extraction

It is well known that graphene oxide (GO), a planar nanomaterial, is endowed with the capacity to immobilize short ssRNA via π-π stacking, thus enhancing its stability. However, whether large RNA molecules, such as total RNA, extracted from biological tissues can be protected using GO has not been investigated. It is usually believed that the protection of total RNA by GO is not effective because the lengths of total RNA, which range from a few to thousands of bases, are inclined to undergo desorption due to their complicated structure. Herein, the nanobiological effects of total RNA/GO are first investigated and demonstrate that the total RNA can be harbored on the surface of GO, thus resulting in a shield effect. This shield effect allows total RNA to highly resist RNase degradation and maintain RNA stability at room temperature up to 4 days, enabling the discovery of GO as the potential next-generation RNase nanoinhibitor. Furthermore, GO can be conjugated to nanomagnetic beads, defined as magnetic graphene oxide, enabling the rapid purification and protection of RNA from animal cells and tissues, whole blood, bacteria, and plant tissue.