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A novel method, monoclonal surface display SELEX (MSD-SELEX), has been designed for simple, rapid, efficient, and cost-effective enrichment and identification of aptamers from a library of monoclonal DNA-displaying beads produced via highly parallel single-molecule emulsion PCR. The approach was successfully applied for the identification of high-affinity aptamers that bind specifically to different types of targets, including cancer biomarker protein EpCAM and small toxin molecule aflatoxin B1. Compared to the conventional sequencing-chemical synthesis-screening work flow, MSD-SELEX avoids large-scale DNA sequencing, expensive and time-consuming DNA synthesis, and labor-intensive screening of large populations of candidates, thus offering a new approach for simple, rapid, efficient, and cost-effective aptamer identification for a wide variety of applications.

Because of their ability to greatly enhance the low natural peroxidase activity of hemin, G-quadruplex-based DNAzymes have been widely used as an alternative to peroxidases for many colorimetric, chemiluminescent, or visual detections of metal ions, small molecules, nucleic acids, proteins, and cancer cells. To obtain G-quadruplex-based DNAzymes with better peroxidase activity, we designed three 81-nt ssDNA libraries containing 25%, 35%, and 45% guanine bases, respectively, at the 45-nt random regions to evolve hemin-binding DNA aptamers using hemin-agarose beads by SELEX (systematic evolution of ligands by exponential enrichment). Some G-rich sequences were obtained after 6 rounds of selection and optimized for stronger binding affinity to hemin and higher peroxidase activity. Our results show that the truncated aptamer [B7]-3-0 folds into compact parallel G-quadruplex structure and exhibits the highest peroxidase activity and strong binding affinity to hemin with 29 ± 4 nM of K(d). It was found that the core G-motifs sequences with 5'-flanking nucleotides exhibit higher peroxidase activity than those with 3'-flanking nucleotides. The numbers of 5'-flanking nucleotides also influence peroxidase activity. In addition, 2'-O-methyl modification facilitates the self-assembly of parallel G-quadruplex [B7]-3-0 and significantly promotes peroxidase activity. This study identifies a G-quadruplex sequence with peroxidase-like activity higher than any other sequences reported so far, which could be potentially used to improve the analytical performance of a wide variety of peroxidase-based bioassays.

Fluorescence anisotropy (FA) is a reliable and excellent choice for fluorescence sensing. One of the key factors influencing the FA value for any molecule is the molar mass of the molecule being measured. As a result, the FA method with functional nucleic acid aptamers has been limited to macromolecules such as proteins and is generally not applicable for the analysis of small molecules because their molecular masses are relatively too small to produce observable FA value changes. We report here a molecular mass amplifying strategy to construct anisotropy aptamer probes for small molecules. The probe is designed in such a way that only when a target molecule binds to the probe does it activate its binding ability to an anisotropy amplifier (a high molecular mass molecule such as protein), thus significantly increasing the molecular mass and FA value of the probe/target complex. Specifically, a mass amplifying probe (MAP) consists of a targeting aptamer domain against a target molecule and molecular mass amplifying aptamer domain for the amplifier protein. The probe is initially rendered inactive by a small blocking strand partially complementary to both target aptamer and amplifier protein aptamer so that the mass amplifying aptamer domain would not bind to the amplifier protein unless the probe has been activated by the target. In this way, we prepared two probes that constitute a target (ATP and cocaine respectively) aptamer, a thrombin (as the mass amplifier) aptamer, and a fluorophore. Both probes worked well against their corresponding small molecule targets, and the detection limits for ATP and cocaine were 0.5 μM and 0.8 μM, respectively. More importantly, because FA is less affected by environmental interferences, ATP in cell media and cocaine in urine were directly detected without any tedious sample pretreatment. Our results established that our molecular mass amplifying strategy can be used to design aptamer probes for rapid, sensitive, and selective detection of small molecules by means of FA in complex biological samples.

A microfluidic device for performing single copy, emulsion Reverse Transcription Polymerase Chain Reaction (RT-PCR) within agarose droplets is presented. A two-aqueous-inlet emulsion droplet generator was designed and fabricated to produce highly uniform monodisperse picoliter agarose emulsion droplets with RT-PCR reagents in carrier oil. Template RNA or cells were delivered from one inlet with RT-PCR reagents/cell lysis buffer delivered separately from the other. Efficient RNA/cell encapsulation and RT-PCR at the single copy level was achieved in agarose-in-oil droplets, which, after amplification, can be solidified into agarose beads for further analysis. A simple and efficient method to graft primer to the polymer matrix using 5'-acrydite primer was developed to ensure highly efficient trapping of RT-PCR products in agarose. High-throughput single RNA molecule/cell RT-PCR was demonstrated in stochastically diluted solutions. Our results indicate that single-molecule RT-PCR can be efficiently carried out in agarose matrix. Single-cell RT-PCR was successfully performed which showed a clear difference in gene expression level of EpCAM, a cancer biomarker gene, at the single-cell level between different types of cancer cells. This work clearly demonstrates for the first time, single-copy RT-PCR in agarose droplets. We believe this will open up new possibilities for viral RNA detection and single-cell transcription analysis.