Molecular beacon mediated circular strand displacement strategy for constructing a ratiometric electrochemical deoxyribonucleic acid sensor

A novel ratiometric electrochemical sensor for sensitive and selective determination of deoxyribonucleic acid (DNA) had been developed based on signal-on and signal-off strategy. The target DNA hybridized with the loop portion of ferrocene (Fc) labeled hairpin probe immobilized on the gold electrode (GE), the Fc away from the surface of GE and the methylene blue (MB) was attached to an electrode surface by hybridization between hairpin probe and MB labeled primer. Such conformational changes resulted in the oxidation peak current of Fc decreased and that of MB increased, and the changes of dual signals are linear with the concentration of DNA. Furthermore, with the help of strand-displacement polymerization, polymerase catalyzed the extension of the primer and the sequential displacement of the target DNA, which led to the release of target and another polymerization cycle. Thus the circular strand displacement produced the multiplication of the MB confined near the GE surface and Fc got away from the GE surface. Therefore, the recognition of target DNA resulted in both the “signal-off” of Fc and the “signal-on” of MB for dual-signal electrochemical ratiometric readout. The dual signal strategy offered a dramatic enhancement of the stripping response. The dynamic range of the target DNA detection was from 10⁻¹³ to 10⁻⁸ mol L⁻¹ with a detection limit down to 28 fM level. Compared with the single signaling electrochemical sensor, the dual-signaling electrochemical sensing strategy developed in this paper was more selective. It would have important applications in the sensitive and selective electrochemical determination of other small molecules and proteins.     

“Signal-on” Electrogenerated Chemiluminescence Biosensing Method for the Determination of Matrix Metalloproteinase 2

A simple, selective and sensitive “signal-on” electrogenerated chemiluminescence (ECL) biosensing method was developed for matrix metalloproteinase 2 (MMP-2). Ru(bpy)₃²⁺, gold nanoparticles (AuNPs) and Nafion were modified onto glassy carbon electrode (GCE) to form Ru(bpy)₃²⁺/AuNPs/Nafion/GCE as sensitive ECL platform and then ferrocene (Fc) labeled peptide was assembled onto the modified electrode to form ECL biosensing platform. The ECL intensity increased when the ECL biosensing electrode reacted with MMP-2 because of MMP-2-induced cleavage of Fc labeled peptide. The ECL method was applied to determine MMP-2 with detection limit of 0.3 ng/mL and one-step recognition, which is promising for point-of-care test of protease.     

Ultrasensitive electrochemiluminescent aptasensor for ochratoxin A detection with the loop-mediated isothermal amplification

In this study, we for the first time presented an efficient, accurate, rapid, simple and ultrasensitive detection system for small molecule ochratoxin A (OTA) by using the integration of loop-mediated isothermal amplification (LAMP) technique and subsequently direct readout of LAMP amplicons with a signal-on electrochemiluminescent (ECL) system. Firstly, the dsDNA composed by OTA aptamer and its capture DNA were immobilized on the electrode. After the target recognition, the OTA aptamer bond with target OTA and subsequently left off the electrode, which effectively decreased the immobilization amount of OTA aptamer on electrode. Then, the remaining OTA aptamers on the electrode served as inner primer to initiate the LAMP reaction. Interestingly, the LAMP amplification was detected by monitoring the intercalation of DNA-binding Ru(phen)₃²⁺ ECL indictors into newly formed amplicons with a set of integrated electrodes. The ECL indictor Ru(phen)₃²⁺ binding to amplicons caused the reduction of the ECL intensity due to the slow diffusion of Ru(phen)₃²⁺–amplicons complex to the electrode surface. Therefore, the presence of more OTA was expected to lead to the release of more OTA aptamer, which meant less OTA aptamer remained on electrode for producing LAMP amplicons, resulting in less Ru(phen)₃²⁺ interlaced into the formed amplicons within a fixed Ru(phen)₃²⁺ amount with an obviously increased ECL signal input. As a result, a detection limit as low as 10 fM for OTA was achieved. The aptasensor also has good reproducibility and stability.     

Simple and label-free electrochemical assay for signal-on DNA hybridization directly at undecorated graphene oxide

Exploring graphene oxide (GO), DNA hybridization detection usually relies on either GO decoration or DNA sequences labeling. The former endows GO with desired chemical, optical, and biological properties. The latter adopts labeled molecules to indicate hybridization. In the present work, we propose a simple, label-free DNA assay using undecorated GO directly as the sensing platform. GO is anchored on diazonium functionalized electrode through electrostatic attraction, hydrogen bonding or epoxy ring-opening. The π–π stacking interaction between hexagonal cells of GO and DNA base rings facilitates DNA immobilization. The adsorbed DNA sequence is specially designed with two parts, including immobilization sequence and probe sequence. In the absence of target, the two sequences lie nearly flat on GO platform. In the presence of target, probe hybridizes with it to form double helix DNA, which ‘stands’ on GO. While the immobilization sequence part remains ‘lying’ on GO surface. Hence, DNA hybridization induces GO interfacial property changes, including negative charge and conformational transition from ‘lying’ ssDNA to ‘standing’ dsDNA. These changes are monitored by electrochemical impedance spectroscopy and adopted as the analytical signal. This strategy eliminates the requirement for GO decoration or DNA labeling, representing a comparatively simple and effective way. Finally, the principle is applied to the detection of conserved sequence of the human immunodeficiency virus 1 pol gene fragment. The dynamic detection range is from 1.0 × 10⁻¹² to 1.0 × 10⁻⁶ M with detection limit of 1.1 × 10⁻¹³ M with 3σ. And the sequences with double- or four-base mismatched are readily distinguishable. In addition, this strategy may hold great promise for potential applications from DNA biosensing to nanostructure framework construction based on the versatile DNA self-assembly.     

A label-free and signal-on supersandwich fluorescent platform for Hg sensing

A label-free supersandwich fluorescent assay was demonstrated for the first time by taking Hg²⁺ as a detection candidate. The principle of the proposed supersandwich fluorescent platform is based on the formation of supersandwich structure by T-Hg²⁺-T coordination and the fluorescence enhancement of the intercalated Genefinder (GF) in double strand DNA (dsDNA). Such supersandwich fluorescent DNA sensor exhibits a linear range of 10–300 nM for the detection of Hg²⁺, with a detection limit of 2.5 nM on the basis of the 3σ/slope (σ represents the standard deviation of the blank samples), which is well below the permit of the U.S. Environmental Protection Agency (<10 nM). The detection can be fulfilled in less than 10 min. The proposed mix-and-detect fluorescent platform exhibits excellent sensitivity, selectivity, and convenient manipulation. The assay was successfully used to detect Hg²⁺ in the lake water samples, which suggested its potential in practical samples.     

Selective, peroxidase substrate based “signal-on” colorimetric assay for the detection of chromium (VI)

Due to the potentially adverse effects of the chromium (VI) on the human health and also on the environment, the quantitative determination of Cr(VI) is of particular interest. This work herein reports a facile, selective and rapid colorimetric determination of Cr(VI) based on the peroxidase substrate-2,2′-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS) as the color developing agent. ABTS, which was usually acted as peroxidase substrate for the enzyme linked immunosorbent assay, is used here for the first time to fabricate the “signal-on” colorimetric Assay for Cr(VI). The ABTS was chosen instead of the commonly used 1,5-diphenylcarbazide (DPC) due to its good solubility, stability, sensitivity and low background. This method provided a convenient colorimetric detection of Cr(VI) with a wider linear range from 8.33 μg L⁻¹ to 1.25 mg L⁻¹ by recording the absorption spectra at the wavelength of 419 nm and a low detection limit of 7.87 μg L⁻¹. In addition, the entire detection takes less than 10 min.     

A new signal-on method for the detection of protein based on binding-induced strategy and photoinduced electron transfer between Ag nanoclusters and split G-quadruplex-hemin complexes

Proteins play important roles in biological and cellular processes. The levels of proteins can be useful biomarkers for cellular events or disease diagnosis, thus the method for sensitive and selective detection of proteins is imperative to proteins express, study, and clinical diagnosis. Herein, we report a “signal-on” platform for the assay of protein based on binding-induced strategy and photoinduced electron transfer between Ag nanoclusters and split G-quadruplex-hemin complexes. By using biotin as the affinity ligand, this simple protocol could sensitively detect streptavidin with a detection limit down to 10 pM. With the use of an antibody as the affinity ligand, a method for homogeneous fluorescence detection of Prostate Specific Antigen (PSA) was also proposed with a detection limit of 10 pM. The one-step and wash-free assay showed good selectivity. Its high sensitivity, acceptable accuracy, and satisfactory versatility of analytes led to various applications in bioanalysis.