Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor protein is the cause of familial VHL disease and sporadic kidney cancer. The VHL gene product (pVHL) is a component of an E3 ubiquitin ligase complex that targets the hypoxia-inducible factor (HIF) 1 and 2 ␣ subunits for polyubiquitylation. This process is dependent on the hydroxylation of conserved proline residues on the ␣ subunits of HIF-1/2 in the presence of oxygen. In our effort to identify orphan HIF-like proteins in the data base that are potential targets of the pVHL complex, we report multiple splice variants of the human HIF-3␣ locus as follows: hHIF-3␣1, hHIF-3␣2 (also referred to as hIPAS; human inhibitory PAS domain protein), hHIF-3␣3, hHIF-3␣4, hHIF-3␣5, and hHIF-3␣6. We demonstrate that the common oxygen-dependent degradation domain of hHIF-3␣1-3 splice variants is targeted for ubiquitylation by the pVHL complex in vitro and in vivo. This activity is enhanced in the presence of prolyl hydroxylase and is dependent on a proline residue at position 490. Furthermore, the ubiquitin conjugation occurs on lysine residues at position 465 and 568 within the oxygen-dependent degradation domain. These results demonstrate additional targets of the pVHL complex and suggest a growing complexity in the regulation of hypoxia-inducible genes by the HIF family of transcription factors.

A highly selective electrogenerated chemiluminescence (ECL) biosensor for the detection of target single-strand DNA (ss-DNA) was developed using hairpin DNA as the recognition element and ruthenium complex as the signal-producing compound. The ECL-based DNA biosensor was fabricated by self-assembling the ECL probe of thiolated hairpin DNA tagged with ruthenium complex on the surface of a gold electrode. In the absence of target ss-DNA, the ECL probe immobilized on the surface of the electrode was in the folded configuration in which its termini were held in close proximity to the electrode, and thus a strong ECL signal could be generated. In the presence of target ss-DNA, a stem-loop of the ECL probe on the electrode was converted into a linear double-helix configuration due to hybridization, resulting in the tag moving away from the electrode surface, which in turn decreased the ECL signal. The ECL intensity of the DNA biosensor generated a "switch off" mode, which decreased with an increase of the concentration of target DNA, and a detection limit of 9 x 10(-11) M complementary target ss-DNA was achieved. Single mismatched target ss-DNA was effectively discriminated from complementary target ss-DNA. The effect of different loop lengths of the hairpin DNA on the selectivity of the ECL DNA biosensor has been investigated. This work demonstrated that the sensitivity and specificity of an ECL DNA biosensor could be greatly improved using a hairpin DNA species which has an appropriate stem and loop length as the recognition element.

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Organic nanoparticles (NPs) from donor-acceptor based coumarin derivatives, 6-[4-(N,N-diphenylamino)phenyl]-3-ethoxycarbonyl coumarin (DPA-CM), with an average size of 5.82 nm, were synthesized by a facile reprecipitation method using water as a poor solvent and tetrahydrofuran as a good solvent. Red-shifted absorption, blue-shifted photoluminescence emission, and aggregation-induced enhanced electrochemiluminescence (ECL) emission were observed for the DPA-CM NPs in aqueous solution compared with the original DPA-CM in organic solution. The aggregation-induced enhanced ECL emission is ascribed to the combined effects of the small size of the DPA-CM NPs, the restricted conformational relaxation in the NPs, and the good stability of the cationic radical of DPA-CM. A strong and stable ECL emission is obtained at the DPA-CM NPs modified glassy carbon electrode in the presence of tri-n-propylamine, and the ECL intensity of the DPA-CM NPs modified electrode is quenched linearly in the range of 0.05-50 μM with detection limit of 0.04, 0.2, and 0.4 μM for ascorbic acid, uric acid, and dopamine, respectively. This work shows an example of donor-acceptor based organic NPs as ECL emitters and their analytical applications to monitor biomolecules.

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An ultrasensitive electrogenerated chemiluminescence peptide-based (ECL-PB) method for the determination of cardiac troponin I (TnI) incorporating amplification of signal reagent-encapsulated liposome was reported for the first time. A synthesized short linear specific binding peptide (FYSHSFHENWPSK) was employed as a molecular recognition element for TnI, which was a reliable biomarker for detecting cardiac injury. Liposomes assembled using a standard sonication procedure were designed as the carrier of ECL signal reagents [bis(2,2'-bipyridine)-4,4'-dicarboxybipyridine ruthenium-di(N-succinimidyl ester) bis(hexafluorophosphate)] for signal amplification. The magnetic capture peptides for the enrichment of the target protein and magnetic separation were synthesized by covalently attaching the peptides to the surface of magnetic beads via an acylation reaction, and the liposome peptides were synthesized by covalently attaching the peptides to the signal reagent-encapsulated liposomes. In the presence of TnI, sandwich-type conjugates were generated in incubation of the magnetic capture peptides and the liposome peptides. After a magnetic separation, the sandwich-type conjugates were treated with ethanol and, thus, a great number of the ECL reagents were released and measured by the ECL method at a bare glassy carbon electrode with a potential pulse of +1.15 V versus Ag/AgCl in the presence of tri-n-propylamine. The increased ECL intensity has good linearity with the logarithm of the TnI concentration in the range from 10 pg/mL to 5.0 ng/mL, with an extremely low detection limit of 4.5 pg/mL. The proposed ECL-PB method was successfully applied to the detection of TnI in human serum samples. This work demonstrated that the employment of the magnetic capture peptides for the enrichment of the target proteins and magnetic separation and the liposome peptides for the signal amplification and polyvalent binding motifs may open a new door to ultrasensitive detection of proteins in clinical analyses.