Electrochemical Aptameric Recognition System for a Sensitive Protein Assay Based on Specific Target Binding-Induced Rolling Circle Amplification

A reusable aptameric recognition system was described for the electrochemical detection of the protein PDGF-BB based on the target binding-induced rolling circle amplification (RCA). A complementary DNA (CDNA), linear padlock probe, and primer probe were utilized to introduce a RCA process into the aptamer−target binding event while a new aptamer was elegantly designed via lengthening the original aptamer by the complement to the CDNA. The aptameric sensing system facilitates the integration of multiple functional elements into a signaling scheme: a unique electrochemical technique, an attractive RCA process, reversible DNA hybridization, and desirable aptameric target recognition. This RCA-based electrochemical recognition system not only exhibits excellent performance (e.g., a detection limit of 6.3 × 10−11 M, a linear dynamic range of 2 orders of magnitude, high specificity, and satisfactory repeatability) but also overcomes the limitations associated with conventional aptameric biosensors (e.g., dependence of signaling target binding on specific aptamer sequence or requirement of sandwich assays for two or more binding sites per target molecule). A recovery test demonstrated the feasibility of the developed target protein assay. Given the attractive characteristics, this aptameric recognition platform is expected to be a candidate for the detection of proteins and other ligands of interest in both fundamental and applied research

Cooperative Amplification-Based Electrochemical Sensor for the Zeptomole Detection of Nucleic Acids

In this work, we developed a multiple-amplification-based electrochemical sensor for ultrasensitive detection of nucleic acids using a disease-related sequence of the p53 gene as the model target. A capture probe (CP) with a hairpin structure is immobilized on the electrode surface via thiol–gold bonding, while its stem is designed to contain a restriction site for <i>Eco</i>RI. In the absence of target DNA, the probe keeps a closed conformation and forms a cleavable region. After treatment with <i>Eco</i>RI, the target binding portion (loop) plus the biotin tag can be peeled off, suppressing the background current. In contrast, the CP is opened by the target hybridization, deforming the restriction site and forcing the biotin tag away from the electrode. On the basis of the biotin–streptavidin complexation, gold nanoparticles (GNPs) modified with a large number of ferrocene-signaling probes (Fc-SPs) are captured by the resulting interface, producing an amplified electrochemical signal due to the GNP-based enrichment of redox-active moieties. Furthermore, Fc tags can be dragged in close proximity to the electrode surface via hybridization between the signaling probes and the CP residues after <i>Eco</i>RI treatment, facilitating interfacial electron transfer and further enhancing the signal. With combination of these factors, the present system is demonstrated to achieve an ultrahigh sensitivity of zeptomole level and a wide dynamic response range of over 7 orders of magnitude

Rolling Circle Amplification Combined with Gold Nanoparticle Aggregates for Highly Sensitive Identification of Single-Nucleotide Polymorphisms

A highly sensitive and specific colorimetry-based rolling circle amplification (RCA) assay method for single-nucleotide polymorphism genotyping has been developed. A circular template is generated by ligation upon the recognition of a point mutation on DNA targets. An RCA amplification is then initiated using the circular template in the presence of Phi29 polymerase. The RCA product can be digested by a restricting endonuclease, and the cleaved DNA fragments can mediate the aggregation of gold nanoparticle-tagged DNA probes. This causes a colorimetric change of the solution as the indicator of the mutation occurrence, which can be detected using UV−vis spectroscopy or viewed by naked eyes. On the basis of the high amplification efficiency of Phi29 polymerase, a mutated target of ∼70 fM can be detected in this assay. In addition, the protection of the circle template using phosphorothioated nucleotides allows the digestion reaction to be performed simultaneously in RCA. Moreover, DNA ligase offers high fidelity in distinguishing the mismatched bases at the ligation site, resulting in positive detection of mutant targets even when the ratio of the wild-type to the mutant is 10 000:1. The developed RCA-based colorimetric detection scheme was demonstrated for SNP typing of β-thalassemia gene at position −28 in genomic DNA

Acetylcholinesterase Liquid Crystal Biosensor Based on Modulated Growth of Gold Nanoparticles for Amplified Detection of Acetylcholine and Inhibitor

A novel acetylcholinesterase (AChE) liquid crystal (LC) biosensor based on enzymatic growth of gold nanoparticles (Au NPs) has been developed for amplified detection of acetylcholine (ACh) and AChE inhibitor. In this method, AChE mediates the hydrolysis of acetylthiocholine (ATCl) to form thiocholine, and the latter further reduces AuCl₄^– to Au NPs without Au nanoseeds. This process, termed biometallization, leads to a great enhancement in the optical signal of the LC biosensor due to the large size of Au NPs, which can greatly disrupt the orientational arrangement of LCs. On the other hand, the hydrolysis of ATCl is inhibited in the presence of ACh or organophosphate pesticides (OPs, a AChE inhibitor), which will decrease the catalytic growth of Au NPs and, as a result, reduce the orientational response of LCs. On the basis of such an inhibition mechanism, the AChE LC biosensor can be used as an effective way to realize the detection of ACh and AChE inhibitors. The results showed that the AChE LC biosensor was highly sensitive to ACh with a detection limit of 15 μmol/L and OPs with a detection limit of 0.3 nmol/L. This study provides a simple and sensitive AChE LC biosensing approach and offers effective signal enhanced strategies for the development of enzyme LC biosensors

Rhodium-Catalyzed Asymmetric Cyclization/Addition Reactions of 1,6-Enynes and Oxa/Azabenzonorbornadienes

A mild, efficient, and novel rhodium catalyzed asymmetric cyclization–addition domino reaction of oxa/azabenzonorbornadienes and 1,6-enynes is documented. Through the use of a [Rh­(COD)₂]­BF₄-(<i>R</i>)-An-SDP catalytic system, highly enantioenriched cyclization–addition products were obtained in good yields and with excellent enantioselectivities

NRS frequency shift comparison between MPA - ME mixed SAM and MPA modifying sensors.

<p><i>Sj</i>Ag and <i>Sj</i>Ab dilution ratios are 1∶5 and 1∶100 respectively. The frequency shift value using the sensor modified by MPA and ME hybrid films normalized to a blank (negative) NRS sample was significantly lower (120 Hz) than that of the sensor modified by MPA film and normalized to the NRS sample (156 Hz, with BSA used alone as a blocking reagent). However, the frequency shift values of the two sensors further declines to 41 Hz and 78 Hz, respectively, with the use of NRS/BSA mixture as blocking reagent.</p

Inhibitory Effect of Target Binding on Hairpin Aptamer Sticky-End Pairing-Induced Gold Nanoparticle Assembly for Light-up Colorimetric Protein Assay

Gold nanoparticles (GNPs) possessing strong distance-dependent optical properties and high extinction coefficients have emerged as important colorimetric materials. Almost all colorimetric studies are based on two working mechanisms: sandwich cross-linking and non-cross-linking systems. In the present study, a new working mechanism, hairpin sticky-end pairing-induced GNP assembly, is introduced based on the discovery of unique aggregation behavior of aptamer-functionalized GNPs. The salt-induced aggregation of oligonucleotide probe-modified GNPs can readily occur due to the sticky-end pairing effect while addition of target molecules favors the formation of the hairpin structure of probe sequences and substantially inhibits the nanoparticle assembly. Along this line, we developed a proof-of-concept colorimetric homogeneous assay using immunoglobulin E (IgE) as an analyte model via transforming a commonly designed “light-down” colorimetric biosensor into a “light-up” one. From the point of view of both conformational transition of aptamer and steric bulk, oligonucleotide−GNPs display an additional stability upon binding to target molecules. The assay showed an extremely high sensitivity from both naked eye observations and absorbance measurements. Compared with almost all existing IgE sensing strategies, the proposed colorimetric system possesses a substantially improved analytical performance. Investigating the assembly behavior of hairpin aptamer-modified GNPs could offer new insight into the dependence of the GNP properties on the structure switching and open a new way to design signaling probes and develop colorimetric assay schemes

Label-Free Liquid Crystal Biosensor Based on Specific Oligonucleotide Probes for Heavy Metal Ions

In this study, to enhance the capability of metal ions disturbing the orientation of liquid crystals (LCs), we designed a new label-free LC biosensor for the highly selective and sensitive detection of heavy metal ions. This strategy makes use of the target-induced DNA conformational change to enhance the disruption of target molecules for the orientation of LC leading to an amplified optical signal. The Hg2+ ion, which possesses a unique property to bind specifically to two DNA thymine (T) bases, is used as a model heavy metal ion. In the presence of Hg2+, the specific oligonucleotide probes form a conformational reorganization of the oligonucleotide probes from hairpin structure to duplex-like complexes. The duplex-like complexes are then bound on the triethoxysilylbutyraldehyde/N,N-dimethyl-N-octadecyl (3-aminopropyl) trimethoxysilyl chloride (TEA/DMOAP)-coated substrate modified with capture probes, which can greatly distort the orientational profile of LC, making the optical image of LC cell birefringent as a result. The optical signal of LC sensor has a visible change at the Hg2+ concentration of low to 0.1 nM, showing good detection sensitivity. The cost-effective LC sensing method can translate the concentration signal of heavy metal ions in solution into the presence of DNA duplexes and is expected to be a sensitive detection platform for heavy metal ions and other small molecule monitors