Xiaolei Zuo

In this communication, we report a highly generalizable strategy, target-responsive electrochemical aptamer switch (TREAS) for the development of aptamer-based biosensors. In a typical TREAS design for ATP detection, the aptamer oligonucleotide dually labeled with thiol and ferrocene groups is hybridized with its complementary strand, and the thiolated duplex is self-assembled on a gold electrode. This duplex is responsive to the target ATP, which liberates the complementary strand and forms the aptamer−target complex. The electroactive ferrocene moiety, which is distal to the electrode surface in the absence of ATP, is moved to the proximal position during the binding-induced structural transition. This binding turns on the electron transfer and leads to measurable electrochemical signals for quantification of ATP. We then demonstrate that TREAS is a signal-on, reagentless sensor that can selectively detect ATP and features both generalizability and simplicity in design.

We have demonstrated a novel sensing strategy employing single-stranded probe DNA, unmodified gold nanoparticles, and a positively charged, water-soluble conjugated polyelectrolyte to detect a broad range of targets including nucleic acid (DNA) sequences, proteins, small molecules, and inorganic ions. This nearly "universal" biosensor approach is based on the observation that, while the conjugated polyelectrolyte specifically inhibits the ability of single-stranded DNA to prevent the aggregation of gold-nanoparticles, no such inhibition is observed with double-stranded or otherwise "folded" DNA structures. Colorimetric assays employing this mechanism for the detection of hybridization are sensitive and convenient--picomolar concentrations of target DNA are readily detected with the naked eye, and the sensor works even when challenged with complex sample matrices such as blood serum. Likewise, by employing the binding-induced folding or association of aptamers we have generalized the approach to the specific and convenient detection of proteins, small molecules, and inorganic ions. Finally, this new biosensor approach is quite straightforward and can be completed in minutes without significant equipment or training overhead.

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.

A limitation of many traditional approaches to the detection of specific oligonucleotide sequences, such as molecular beacons, is that each target strand hybridizes with (and thus activates) only a single copy of the relevant probe sequence. This 1:1 hybridization ratio limits the gain of most approaches and thus their sensitivity. Here we demonstrate a nuclease-amplified DNA detection scheme in which exonuclease III is used to "recycle" target molecules, thus leading to greatly improved sensitivity relative to, for example, traditional molecular beacons without any significant restriction in the choice of target sequences. The exonuclease-amplified assay can detect target DNA at concentrations as low as 10 pM when performed at 37 degrees C, which represents a significant improvement over the equivalent molecular beacon alone. Moreover, at 4 degrees C we can obtain a detection limit as low as 20 aM, albeit at the cost of a 24 h incubation period. Finally, our assay can be easily interrogated with the naked eye and is thus amenable to deployment in the developing world, where fluorometric detection is more problematic.

Self-assembled DNA nanostructures with precise sizes allow a programmable "soft lithography" approach to engineer the interface of electrochemical DNA sensors. By using millimeter-sized gold electrodes modified with several types of tetrahedral DNA nanostructures (TDNs) of different sizes, both the kinetics and thermodynamics of DNA hybridization were profoundly affected. Because each DNA probe is anchored on an individual TDN, its lateral spacing and interactions are finely tuned by the TDN size. By simply varying the size of the TDNs, the hybridization time was decreased and the hybridization efficiency was increased. More significantly, the detection limit for DNA detection was tuned over four orders of magnitude with differentially nanostructured electrodes, and achieved attomolar sensitivity with polymeric enzyme amplification.