Xiaolei Zuo

High Specificity, Electrochemical Sandwich Assays Based on Single Aptamer Sequences and Suitable for the Direct Detection of Small-Molecule Targets in Blood and Other Complex Matrices

We herein demonstrate a sandwich assay based on single aptamer sequences is suitable for the direct detection of small molecule targets in blood serum and other complex matrices. By splitting an aptamer into two pieces, we convert a single affinity reagent into a two-component system in which the presence of the target drives formation of a complex comprised of the target and the two halves of the aptamer. To demonstrate the utility of this approach we have used single anticocaine and anti-ATP aptamers to fabricate electrochemical sensors directed against the representative small molecules cocaine and ATP. Both targets are detected at low micromolar concentrations, in seconds, and in a convenient, general, readily reusable, electrochemical format. Moreover, both sensors are selective enough to deploy directly in blood, crude cellular lysates and other complex sample matrices.

Lab in a tube: ultrasensitive detection of microRNAs at the single-cell level and in breast cancer patients using quadratic isothermal amplification.

Through rational design of a functional molecular probe with high sequence specificity that takes advantage of sensitive isothermal amplification with simple operation, we developed a one-pot hairpin-mediated quadratic enzymatic amplification strategy for microRNA (miRNA) detection. Our method exhibits ultrahigh sensitivity toward miR-21 with detection limits of 10 fM at 37 °C and 1 aM at 4 °C, which corresponds to nine strands of miR-21 in a 15 μL sample, and it is capable of distinguishing among miRNA family members. More importantly, the proposed approach is also sensitive and selective when applied to crude extractions from MCF-7 and PC3 cell lines and even patient tissues from intraductal carcinoma and invasive ductal carcinoma of the breast.

Hybridization Chain Reaction Amplification of MicroRNA Detection with a Tetrahedral DNA Nanostructure-Based Electrochemical Biosensor

There remains a great challenge in the sensitive detection of microRNA because of the short length and low abundance of microRNAs in cells. Here, we have demonstrated an ultrasensitive detection platform for microRNA by combining the tetrahedral DNA nanostructure probes and hybridization chain reaction (HCR) amplification. The detection limits for DNA and microRNA are 100 aM and 10 aM (corresponding to 600 microRNAs in a 100 μL sample), respectively. Compared to the widely used supersandwich amplification, the detection limits are improved by 3 orders of magnitude. The uncontrolled surface immobilization and consumption of target molecules that limit the amplification efficiency of supersandwich are eliminated in our platform. Taking advantage of DNA nanotechnology, we employed three-dimensional tetrahedral DNA nanostructure as the scaffold to immobilize DNA recognition probes to increase the reactivity and accessibility, while DNA nanowire tentacles are used for efficient signal amplification by capturing multiple catalytic enzymes in a highly ordered way. The synergetic effect of DNA tetrahedron and nanowire tentacles have proven to greatly improve sensitivity for both DNA and microRNA detection.

Graphene Oxide-Facilitated Electron Transfer of Metalloproteins at Electrode Surfaces

Graphene is a particularly useful nanomaterial that has shown great promise in nanoelectronics. Because of the ultrahigh electron mobility of graphene and its unique surface properties such as one-atom thickness and irreversible protein adsorption at surfaces, graphene-based materials might serve as an ideal platform for accommodating proteins and facilitating protein electron transfer. In this work, we demonstrate that graphene oxide (GO) supports the efficient electrical wiring the redox centers of several heme-containing metalloproteins (cytochrome c, myoglobin, and horseradish peroxidase) to the electrode. Importantly, proteins retain their structural intactness and biological activity upon forming mixtures with GO. These important features imply the promising applications of GO/protein complexes in the development of biosensors and biofuel cells.

Flexible Carbon Nanotube-Polymer Composite Films with High Conductivity and Superhydrophobicity Made by Solution Process

CNT/Nafion nanocomposite film made by solution process exhibits high conductivity and superhydrophobicity. The highest water contact angle reaches 165.3 +/- 1.9 degrees. The wettability of the film can be controlled by simply varying the filtering rate and the content ratio of Nafion to CNT. We also develop a novel optical method to directly observe the air-solid-liquid interface for the first time. The extraordinary mechanical strength provided by the polymer helps the film retain its conductivity and superhydrobicity after 1000 bending cycles.

Recent Development of Sandwich Assay Based on the Nanobiotechnologies for Proteins, Nucleic Acids, Small Molecules, and Ions

Nanobiotechnologies for Proteins, Nucleic Acids, Small Molecules, and Ions Juwen Shen, Yuebin Li,†,§,⊥ Haoshuang Gu, Fan Xia,*,† and Xiaolei Zuo*,‡ †Key Laboratory for Large-Format Battery Materials and System, Ministry of Education, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology (HUST), Wuhan 430074, China ‡Division of Physical Biology and Bioimaging Center, Shanghai Synchrotron Radiation Facility, Shanghai Institute of Applied Physics, Chinese Academy of Science, Shanghai 201800, China Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Faculty of Physical and Electronic Sciences, Hubei University, Wuhan 430062, China Key Laboratory of Systems Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

Comparing the Properties of Electrochemical-Based DNA Sensors Employing Different Redox Tags

Many electrochemical biosensor approaches developed in recent years utilize redox-labeled (most commonly methylene blue or ferrocene) oligonucleotide probes site-specifically attached to an interrogating electrode. Sensors in this class have been reported that employ a range of probe architectures, including single- and double-stranded DNA, more complex DNA structures, DNA and RNA aptamers, and, most recently, DNA-small molecule chimeras. Signaling in this class of sensors is generally predicated on binding-induced changes in the efficiency with which the covalently attached redox label transfers electrons with the interrogating electrode. Here we have investigated how the properties of the redox tag affect the performance of such sensors. Specifically, we compare the differences in signaling and stability of electrochemical DNA sensors (E-DNA sensors) fabricated using either ferrocene or methylene blue as the signaling redox moiety. We find that while both tags support efficient E-DNA signaling, ferrocene produces slightly improved signal gain and target affinity. These small advantages, however, come at a potentially significant price: the ferrocene-based sensors are far less stable than their methylene blue counterparts, particularly with regards to stability to long-term storage, repeated electrochemical interrogations, repeated sensing/regeneration iterations, and employment in complex sample matrices such as blood serum.

Gold nanoparticle-decorated MoS2 nanosheets for simultaneous detection of ascorbic acid, dopamine and uric acid

An electrochemical sensor has been developed for simultaneous detection of dopamine (DA), uric acid (UA) and ascorbic acid (AA) based on a gold nanoparticle-decorated MoS2 nanocomposite (AuNPs@MoS2) modified electrode. The AuNPs@MoS2 nanocomposite has been synthesized by electrodeposition of AuNPs on the MoS2 nanosheets, which possesses better properties than pure AuNPs and MoS2. The AuNPs@MoS2 film modified electrode showed excellent electrocatalytic activity toward the oxidation of AA, DA and UA with three well-resolved oxidation peaks. The peak separation of AA–DA, DA–UA and AA–UA is 151 mV, 137 mV and 288 mV, respectively, which permits the modified electrode to individually or simultaneously analyze AA, DA and UA by differential pulse voltammetry (DPV). Under optimum conditions, the AuNPs@MoS2 modified electrode exhibits linear response toward AA, DA and UA in the range of 50–100 000 μM, 0.05–30 μM and 50–40 000 μM, respectively. Moreover, the MoS2-based modified electrode was successfully employed to determine DA in human serum samples with satisfactory results.

Creating SERS Hot Spots on MoS2 Nanosheets with in Situ Grown Gold Nanoparticles

Herein, a reliable surface-enhanced Raman scattering (SERS)-active substrate has been prepared by synthesizing gold nanoparticles (AuNPs)-decorated MoS2 nanocomposite. The AuNPs grew in situ on the surface of MoS2 nanosheet to form efficient SERS hot spots by a spontaneous redox reaction with tetrachloroauric acid (HAuCl4) without any reducing agent. The morphologies of MoS2 and AuNPs-decorated MoS2 nanosheet were characterized by TEM, HRTEM, and AFM. The formation of hot spots greatly depended on the ratio of MoS2 and HAuCl4. When the concentration of HAuCl4 was 2.4 mM, the as-prepared AuNPs@MoS2-3 nanocomposite exhibited a high-quality SERS activity toward probe molecule due to the generated hot spots. The spot-to-spot SERS signals showed that the relative standard deviation (RSD) in the intensity of the main Raman vibration modes (1362, 1511, and 1652 cm(-1)) of Rhodamine 6G were about 20%, which displayed good uniformity and reproducibility. The AuNPs@MoS2-based substrate was reliable, sensitive, and reproducible, which showed great potential to be an excellent SERS substrate for biological and chemical detection.

Electrochemical detection of nucleic acids, proteins, small molecules and cells using a DNA-nanostructure-based universal biosensing platform

The occurrence and prognosis of many complex diseases, such as cancers, is associated with the variation of various molecules, including DNA at the genetic level, RNA at the regulatory level, proteins at the functional level and small molecules at the metabolic level (defined collectively as multilevel molecules). Thus it is highly desirable to develop a single platform for detecting multilevel biomarkers for early-stage diagnosis. Here we report a protocol on DNA-nanostructure-based programmable engineering of the biomolecular recognition interface, which provides a universal electrochemical biosensing platform for the ultrasensitive detection of nucleic acids (DNA/RNA), proteins, small molecules and whole cells. The protocol starts with the synthesis of a series of differentially sized, self-assembled tetrahedral DNA nanostructures (TDNs) with site-specifically modified thiol groups that can be readily anchored on the surface of a gold electrode with high reproducibility. By exploiting the rigid structure, nanoscale addressability and versatile functionality of TDNs, one can tailor the type of biomolecular probes appended on individual TDNs for the detection of specific molecules of interest. Target binding occurring on the gold surface patterned with TDNs is quantitatively translated into electrochemical signals via a coupled enzyme-based catalytic process. This uses a sandwich assay strategy in which biotinylated reporter probes recognize TDN-bound target biomolecules, which then allow binding of horseradish-peroxidase-conjugated avidin (avidin–HRP). Hydrogen peroxide (H2O2) is then reduced by avidin–HRP in the presence of TMB (3,3′,5,5′-tetramethylbenzidine) to generate a quantitative electrochemical signal. The time range for the entire protocol is ∼1 d, whereas the detection process takes ∼30 min to 3 h.