Xiaoteng Luo

Conformation-dependent exonuclease III activity mediated by metal ions reshuffling on thymine-rich DNA duplexes for an ultrasensitive electrochemical method for Hg2+ detection.

Hg(2+) is known to bind very strongly with T-T mismatches in DNA duplexes to form T-Hg(2+)-T base pairs, the structure of which is stabilized by covalent N-Hg bonds and exhibits bonding strength higher than hydrogen bonds. In this work, we exploit exonuclease III (Exo III) activity on DNA hybrids containing T-Hg(2+)-T base pairs and our experiments show that Hg(2+) ions could intentionally trigger the activity of Exo III toward a designed thymine-rich DNA oligonucleotide (e-T-rich probe) by the conformational change of the probe. Our sensing strategy utilizes this conformation-dependent activity of Exo III, which is controlled through the cyclical shuffling of Hg(2+) ions between the solution phase and the solid DNA hybrid. This interesting attribute has led to the development of an ultrasensitive detection platform for Hg(2+) ions with a detection limit of 0.2 nM and a total assay time within minutes. This simple detection strategy could be used for the detection of other metal ions which exhibit specific interactions with natural or synthetic bases.

Organic Electrochemical Transistors Integrated in Flexible Microfluidic Systems and Used for Label‐Free DNA Sensing

enzyme sensors, [ 9 ] DNA sensors, [ 10 ] dopamine sensor, [ 11 ] and cell-based biosensors. [ 12 ] A transistor-based sensor is the combination of a sensor and an amplifi er since a small potential change at an interface can induce a substantial variation of the channel current. [ 16–18 ] Therefore such devices are highly sensitive and potentially low cost. More importantly, OECT can be easily miniaturized and fabricated on fl exible substrates, which is essential for some applications in living systems. OECTs have been integrated in microfl uidic channels and used as ion sensors and enzyme sensors. [ 5 , 6 , 9 ] As for fl exible devices, OECTs were fabricated on fi bers and showed excellent transistor performance. [ 19 , 20 ] However, the application of the fi ber-supported devices in biosensors is limited by solid electrolytes used in the devices. Therefore, we study the OECT integrated in a fl exible microfl uidic system and explore its applications in biosensors, such as DNA sensors. Nucleic acid diagnostics has attracted much interest due to its great scientifi c and economic importance, and it has signifi cant applications in gene expression monitoring, viral and bacterial identifi cation, biowarfare and bioterrorism agents detecting, and clinical medicine. [ 21 ] Besides the traditional technique that is based on the confocal fl uorescence microscope, several different label-free technologies have been developed for the analysis of DNA microarrays, including atomic force microscopy, [ 22 ] electrochemical detection, [ 23 ] surface vibration spectroscopy, [ 24 ] scanning Kelvin probe microscopy (SKPM), [ 25 ]

Immobilization-free sequence-specific electrochemical detection of DNA using ferrocene-labeled peptide nucleic acid.

An electrochemical method for sequence-specific detection of DNA without solid-phase probe immobilization is reported. This detection scheme starts with a solution-phase hybridization of ferrocene-labeled peptide nucleic acid (Fc-PNA) and its complementary DNA (cDNA) sequence, followed by the electrochemical transduction of Fc-PNA-DNA hybrid on indium tin oxide (ITO)-based substrates. On the bare ITO electrode, the negatively charged Fc-PNA-DNA hybrid exhibits a much reduced electrochemical signal than that of the neutral-charge Fc-PNA. This is attributed to the electrostatic repulsion between the negatively charged ITO surface and the negatively charged DNA, hindering the access of Fc-PNA-DNA to the electrode. On the contrary, when the transduction measurement is done on the ITO electrode coated with a positively charged poly(allylamine hydrochloride) (PAH) layer, the electrostatic attraction between the (+) PAH surface and the (-) Fc-PNA-DNA hybrid leads to a much higher electrochemical signal than that of the Fc-PNA. The measured electrochemical signal is proportional to the amount of cDNA present. In terms of detection sensitivity, the PAH-modified ITO platform was found to be more sensitive (with a detection limit of 40 fmol) than the bare ITO counterpart (with a detection limit of 500 fmol). At elevated temperatures, this method was able to distinguish fully matched target DNA from DNA with partial mismatches. Unpurified PCR amplicons were detected using a similar format with a detection limit down to 4.17 amol. This detection method holds great promise for single-base mismatch detection as well as electrochemistry-based detection of post-PCR products.

Immobilization-free multiplex electrochemical DNA and SNP detection.

A novel electrochemical method for multiplex detection of sequence-specific DNA and single nucleotide polymorphism (SNP) that requires no probe immobilization is reported. The immobilization-free detection of target DNA is realized by the use of a neutrally charged peptide nucleic acid (PNA) probe labeled with an electroactive indicator and a negatively charged ITO electrode. Upon hybridization between the target DNA and the complementary PNA probe, electrostatic repulsion between the negative backbone of the DNA/PNA duplex and the negative surface of the ITO electrode prevents the electroactive indicator from approaching the electrode, resulting in a significantly suppressed electrochemical signal. Multiplex DNA or SNP detection is enabled by using multiple PNA probes with different sequences labeled with different distinguishable electroactive indicators. Due to the immobilization-free nature of this detection scheme, no interference is found between the simultaneous detection of multiple target DNAs or SNPs. This simple and robust immobilization-free multiplex DNA and SNP detection strategy may find applications in a wide range of fields especially in point-of-care testing.

Staining-free gel electrophoresis-based multiplex enzyme assay using DNA and peptide dual-functionalized gold nanoparticles

We report a simple staining‐free gel electrophoresis method to simultaneously probe protease and nuclease. Utilizing gold nanoparticles (Au‐NPs) dual‐functionalized with DNA and peptide, the presence and concentration of nuclease and protease are determined concurrently from the relative position and intensity of the bands in the staining‐free gel electrophoresis. The use of Au‐NPs eliminates the need for staining processes and enables naked eye detection, while a mononucleotide‐mediated approach facilitates the synthesis of DNA/peptide conjugated Au‐NPs and simplifies the operation procedures. Multiplex detection and quantification of DNase I and trypsin are successfully demonstrated.

Yeast surface display-based microfluidic immunoassay

Abstract In this paper, we present a new microfluidic immunoassay platform, which is based on the synergistic combination of the yeast surface display (YSD) technique and the microfluidic technology. Utilizing the YSD technique, antigens specific to the target antibody are displayed on the surface of engineered yeast cells with intracellular fluorescent proteins. The displayed antigens are then used for the detection of the target antibody, with the yeast cells as fluorescent labels. Multiplex immunoassay can be readily realized by using yeast cells expressing different intracellular fluorescent proteins to display different antigens. The implementation of this YSD-based immunoassay on the microfluidic platform eliminates the need for the bulky, complex and expensive flow cytometer. To improve the detection sensitivity and to eliminate the need for pumping, a functionalized micro pillar array (MPA) is incorporated in the microfluidic chip, resulting in a detection limit of 5ng/mL (or 1ng in terms of amount) and enhanced compatibility with practical applications such as clinical biopsy. This new platform has a high potential to be integrated into microfluidic detection systems to enable portable diagnostics in the future.