Zhiyuan Fang

Computational lateral flow biosensor for proteins and small molecules: a new class of strip logic gates.

The first example of strip logic gates (OR and AND functions) for proteins and small molecules has been constructed on the basis of target-induced self-assembly of split aptamer fragments. Using thrombin and ATP as inputs, the corresponding split/integrated aptamers as molecular recognition elements, and gold nanoparticles as a tracer, the output signals can be directly visualized by observing the red bands on the test zones of the strips. The assay is simple, easy to perform, and cost-effective, allowing portable analysis at ambient temperature. The strip logic system is resistant to nonspecific interfering agents and can operate effectively even in human serum samples. Such logic strips hold great promise for application in intelligent point-of-care and in-field diagnostics.

Lateral flow biosensor for DNA extraction-free detection of Salmonella based on aptamer mediated strand displacement amplification.

Convenient and sensitive point-of-care rapid diagnostic tests for food-borne pathogens have been a long-felt need of clinicians. Traditional approaches such as culture-based methods have good sensitivity and specificity, but they tend to be tedious and time-consuming. Herein we present a simple and sensitive aptamer based biosensor for rapid detection of Salmonella enteritidis (S. enteritidis). One of the aptamers specific for the outmembrane of S. enteritidis was used for magnetic bead enrichments. Another aptamer against S. enteritidis was used as a reporter for this pathogen, which was amplified by isothermal strand displacement amplification (SDA) and further detected by a lateral flow biosensor. As low as 10(1) colony forming unit (CFU) of S. enteritidis was detected in this study. Without DNA extraction, the reduced handling and simpler equipment requirement render this assay a simple and rapid alternative to conventional methods.

A sensitive lateral flow biosensor for Escherichia coli O157:H7 detection based on aptamer mediated strand displacement amplification.

Foodborne diseases caused by pathogens are one of the major problems in food safety. Convenient and sensitive point-of-care rapid diagnostic tests for food-borne pathogens have been a long-felt need of clinicians. Commonly used methods for pathogen detection rely on conventional culture-based tests, antibody-based assays and polymerase chain reaction (PCR)-based techniques. These methods are costly, laborious and time-consuming. Herein, we present a simple and sensitive aptamer based biosensor for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7). In this assay, two different aptamers specific for the outmembrane of E. coli O157:H7 were used. One of the aptamers was used for magnetic bead enrichment, and the other was used as a signal reporter for this pathogen, which was amplified by isothermal strand displacement amplification (SDA) and further detected by a lateral flow biosensor. Only the captured aptamers on cell membrane were amplified, limitations of conventional DNA amplification based method such as false-positive can be largely reduced. The generated signals (red bands on the test zone of a lateral flow strip) can be unambiguously read out by the naked eye. As low as 10 colony forming units (CFU) of E. coli O157:H7 were detected in this study. Without DNA extraction, the reduced handling and simpler equipment requirement render this assay a simple and rapid alternative to conventional methods.

An Enhanced Strip Biosensor for Rapid and Sensitive Detection of Histone Methylation

Histone methylation is a crucial epigenetic modification of chromosomes. In this work, we describe an enhanced strip biosensor using oligonucleotide-functionalized gold nanoparticles as an enhancer probe (AuNP-DNA) for rapid and sensitive detection of histone methylation. In conventional strip biosensor, methylated histone is captured on the test zone through the formation of antibody/methylated histone/antibody-labeled AuNP sandwich structures. Whereas, in the enhanced strip biosensor, the AuNPs in the sandwich structures are dual labeled with an antibody and another oligonucleotide (c-DNA). The sequence of the c-DNA is complementary to the oligonucleotide on the enhancer probe. The enhancer probe, AuNP-DNA, hybridizes with the c-DNA on the dual labeled AuNPs, and the color intensity of the red band on the test zone is then enhanced dramatically. The enhanced strip biosensor has been used for the visual detection of trimethylated lysine 9 of histone H3 (H3K9me3) in 20 ng of histone extract from HeLa cells within 15 min. The detection limit is 10-fold and 15-fold lower than the conventional strip biosensor and Western blot, respectively.

An enzyme-free and label-free assay for copper(II) ion detection based on self-assembled DNA concatamers and Sybr Green I

An enzyme-free and label-free fluorescence turn on biosensor for amplified copper(II) ion (Cu(2+)) detection has been constructed based on self-assembled DNA concatamers and Sybr Green I. This assay is simple, inexpensive and sensitive, enabling quantitative detection of as low as 12.8 pM Cu(2+).

A lateral flow biosensor for rapid detection of DNA-binding protein c-jun.

A lateral flow biosensor based on an immuno-chromatographic assay has been developed for the detection of DNA-binding proteins. The biosensor is composed of four parts: a sample pad, a conjugate pad, a strip of nitrocellulose membrane and an absorbent pad. A DNA probe containing a specific protein binding consensus sequence is coated onto gold nanoparticles, while an antibody against the DNA-binding protein is immobilized onto a test zone of the nitrocellulose membrane. The target protein binds to the protein binding DNA sequence that is coated on the gold nanoparticles to form nanoparticle-DNA-protein complexes, and the complexes are then captured by the antibody immobilized on the test zone to form a red line for visual detection of the target protein. This biosensor was successfully applied to a DNA-binding protein, c-jun, and the developed biosensor allows for the rapid detection of down to 0.2 footprint unit of c-jun protein within 10 min. This biosensor was verified using HeLa cells and it visually detected c-jun activity in 100 μg of crude cell lysate protein. The antibody against c-jun used in the biosensor can distinguish c-jun from other nonspecific proteins, with high specificity.

A lateral flow biosensor for the detection of human pluripotent stem cells.

A lateral flow biosensor based on immunoassay has been developed for the detection of human stem cells for the first time. Antibody specific for a human stem cell surface antigen, SSEA-4, is coated onto gold nanoparticles, whereas antibody against another human pluripotent stem cell surface antigen, SSEA-3, is immobilized on the test zone of the NC membrane. Target cells bind to the antibody coated on the gold nanoparticles to form nanoparticles-stem cell complexes, and the complexes are then captured by another antibody immobilized on the test zone to form a red line for visual detection. This biosensor has been successfully applied to human embryonic stem cells and induced pluripotent stem cells. It is capable of detecting a minimum of 10,000 human embryonic stem cells by the naked eye and 7000 cells with a portable strip reader within 20 min. This approach has also shown excellent specificity to distinguish other types of cells. The biosensor shows great promise for specific and handy detection of human pluripotent stem cells.

An autonomous T-rich DNA machine based lateral flow biosensor for amplified visual detection of mercury ions

An autonomous thymine rich DNA machine as an amplification unit was developed for the sensitive detection of mercury ions with high specificity. Combined with a lateral flow biosensor, the amplified signal of Hg2+ can be read out by the naked eye with a detection limit of 5 nM.

A simple aptamer biosensor for Salmonellae enteritidis based on fluorescence-switch signaling graphene oxide

In this communication, we report a rapid and cost-efficient assay for the detection of S. enteritidis. Specific aptamers with fluorescence labeled S. enteritidis are absorbed into graphene oxide, and the fluorescence is quenched owing to the fluorescence quenching ability of graphene oxide. In the presence of S. enteritidis, the aptamer would release from the graphene oxide to obtain a significant fluorescence recovery. This aptasensor can detect as low as 40 CFU mL−1 of S. enteritidis in 30 min, and can fulfill the demand of multiplex detection. The cost-effective, rapid and simple aptasensor offers a promising method for food-borne pathogen monitoring during food processing.

Spectrophotometric and ultrasensitive DNA bioassay by circular-strand displacement polymerization reaction

We demonstrated a new spectrophotometric DNA detection approach based on a circular strand-displacement polymerization reaction for the quantitative detection of sequence specific DNA. In this assay, the hybridization of an immobilized hairpin probe on the microtiter plate, to target DNA, results in a conformational change and leads to a stem separation. A short primer thus anneals with the open stem and triggers a polymerization reaction, allowing a cyclic reaction comprising the release of target DNA and hybridization of the target with the remaining immobilized hairpin probe. Through this cyclical process, a large number of duplex DNA complexes are produced. Finally, the biotin modified duplex DNA products can be detected via the HRP catalyzed substrate 3,3,5,5-tetramethylbenzidine using a spectrophotometer. As a proof of concept, a short DNA sequence (20-nt) related to the South East Asia (SEA) type deletion of α-thalassemia was chosen as the model target. This proposed assay has a very high sensitivity and selectivity with a dynamic response ranging from 0.1 fM to 10 nM and the detection limit was 8 aM. It can be performed within 2 hours, and it can differentiate target SEA DNA from wild-type DNA. By substituting the hairpin probes used in the present work, this assay can be used to detect other subtypes of genetic disorders.