A sensitive, non-damaging electrochemiluminescent aptasensor via a low potential approach at DNA-modified gold electrodes.

Electrochemiluminescence (ECL)-based biosensors are often used in the field of DNA- and protein-assay. Although ruthenium complex-based ECL is sensitive, its high exciting potential may lead to oxidation damage to biomolecules. For the first time, a non-damaging, low potential ECL aptasensor was constructed for bioassay with lysozyme as a model. After a single-stranded anti-lysozyme aptamer was attached to a gold electrode, a double stranded (ds)-DNA formed with its complementary strand. Ru(phen)(3)(2+), as an ECL probe, was intercalated into the ds-DNA. The hybridization of lysozyme with its aptamer led to the dissociation of ds-DNA because of the high stability of the aptamer-lysozyme and therefore the Ru(phen)(3)(2+) intercalated into ds-DNA was released. A low potential ECL was observed at the ds-DNA-modified electrode because ds-DNA was able to preconcentrate tripropylamine (TPA) and acted as the acceptor of the protons released from protonated TPAH(+). While the DNA sequence (anti-lysozyme aptamer) was used as the special recognition element for lysozyme, the formed ds-DNA also provided a micro-environment for low potential ECL. The low potential ECL aptasensor achieved the determination of lysozyme with a detection limit of 0.45 pM. The day-to-day precision (RSDs, n = 5) for the determination of lysozyme was lower than 5%, showing the reliability of the aptasensor. The regeneration of the aptasensor confirmed that the low potential for ECL could decrease oxidation damage to biomolecules. Further, the proposed method was successfully used to analyze diluted egg white sample directly. The protocol exhibited a promising platform for sensitive bioassay and could be further applied for the development of other low potential ECL sensing systems.

The electrochemiluminescence of ruthenium complex/tripropylamine systems at DNA-modified gold electrodes.

The electrochemiluminescence (ECL) behavior of ruthenium complex/tripropylamine (TPA) systems at DNA-modified gold electrode was studied to understand the possible mechanism and to develop new detection platforms. DNA strand, especially double-stranded DNA (ds-DNA), can preconcentrate TPA and acts as the acceptor of the protons released from TPAH(+), therefore the improved ECL emission and the low potential ECL were observed. The intercalation of Ru(phen)(3)(2+) into ds-DNA was confirmed to be a sensitive and label-free DNA-related detection platform. The above results were validated by the analysis of lysozyme using anti-lysozyme aptamer-modified electrode. This work opens a new field by the use of DNA-modified electrode to develop novel sensing platforms, such as low potential ECL biosensors and Ru(phen)(3)(2+) intercalation-based ECL biosensors.

Selective differentiation of indoleacetic acid and indolebutyric acid using colorimetric recognition after Ehrlich reaction.

A simple colorimetric method for the differentiation of indoleacetic acid (IAA) and indolebutyric acid (IBA) in plant samples is described. The color change is based upon the reaction between the auxins and p-(dimethylamino)benzaldehyde (PDAB, Ehrlich reagent) following the electrophilic substitution reaction mechanism at the indole ring. Using their different response to reaction temperature and time, the selective determination of IBA in the presence of IAA is achieved by controlling the incubation time of 40 min at 25 degrees C. The total absorbance of IAA and IBA is determined after they react to PDAB for 150 min at 70 degrees C. The concentration of IAA can then be calculated using the difference between their total absorbance and the calculated absorbance of IBA. The detection limits (3sigma) of IAA and IBA were 0.10 microM and 0.28 microM, respectively. The precisions for five replicate measurements of 10 microM IAA and IBA were less than 5% (RSD). The recovery from mung bean sprout samples varied from 87.5% to 108% for the two auxins. Moreover, the Ehrlich reaction conditions are compatible with the methanol-hydrochloric acid extraction procedure. All of the above results indicate that this protocol provides a rapid, simple, convenient and practical method for detection and differentiation of IAA and IBA. From the color changes of IAA and IBA after Ehrlich reaction, the identification of auxin at the microM level can be achieved even with the naked eye. The method was successfully used to investigate the auxin changes of mung bean sprout during the growth procedure.

Label-free electrochemiluminescent aptasensor with attomolar mass detection limits based on a Ru(phen)(3)(2+)-double-strand DNA composite film electrode.

Precisely known ligand-induced conformation change and complex chemical labeling of the DNA sequence with probe molecules are often needed for the signal generation in most of the previous aptasensors. Herein, a solution to the above problems was reported by the use of the Ru(phen)(3)(2+) intercalated into double strand DNA (ds-DNA) as an electrochemiluminescence (ECL) probe with thrombin as the target. After the antithrombin thiolated aptamer (27-mer) was attached to a gold electrode, ds-DNA structure was formed with its complementary 20-mer single strand DNA. Instead of the chemical modification of the aptamer or target with the probe molecule, Ru(phen)(3)(2+), as the probe, was intercalated into the ds-DNA structure. After thrombin hybridized with its aptamer, the ds-DNA dissociated and the intercalated Ru(phen)(3)(2+) released because of the higher stability of the aptamer-thrombin complex than that of the aptamer-complementary strand hybrid. The difference in ECL intensity with tripropylamine (TPA) as coreactant before and after the hybridization of thrombin and its aptamer was used to quantify thrombin. Besides the increase in the number of probe molecules over the single-site labeling, a ca. 80-fold improvement on the TPA oxidation at the ds-DNA modified electrode was found over the bare gold electrode. With the two amplification factors, the mass detection limits of 0.2 attomolar for thrombin are obtained. Because of the independence of conformational changes, the present method is readily extended to the targets whose aptamers have no specific conformational changes or other DNA-related detection without the need for chemical labeling.