Electrostatic Force Triggering Elastic Condensation of Double-Stranded DNA for High-Performance One-Step Immunoassay.

Current strategies for high-performance immunoassay generally require a sandwich structure for signal amplification. This strategy is limited to multivalent antigens and complicates the detection scheme. Herein we demonstrate a class of simple one-step ultrasensitive immunoassay with the adoption of double-stranded DNA (dsDNA) as "conductive spring" to bridge the electrode and redox-reporter/antibody-receptor comodified gold nanoparticles (AbFc@AuNPs). Upon biorecognition between antigen and antibody, the charge of the AuNPs changes, enhancing the electrostatic interaction between the AuNPs and Au electrode surface, and condensing the dsDNA chain. For the first time, the sensitive response of the electrochemical redox current to the DNA chain length is utilized to achieve an ultrahigh sensitivity down to fM level. Only the primary antibody needed in the recognition interface ensures the one-step immunoreaction works well with monovalent antigens, which ensure this method as a promising general alternative means for fast, high-throughput or point-of-care clinical applications even for very challenging clinically relevant samples.

Light-Up Nonthiolated Aptasensor for Low-Mass, Soluble Amyloid-ß40 Oligomers at High Salt Concentrations.

Herein, a light-up nonthiolated aptasensor was developed for low-mass, soluble amyloid-ß40 oligomers (LS-Aß40-O). Au nanoparticles (AuNP) were employed as colorimetric probes, and the nonthiolated aptamers (Apt) were adsorbed on AuNP surfaces, acting as binding elements for LS-Aß40-O. The aggregation of AuNPs was induced when Apt-modified AuNPs (Apt@AuNPs) were under high-salt conditions. However, upon the addition of LS-Aß40-O into the Apt@AuNP solution, the salt tolerance of the AuNPs was greatly enhanced. Further studies confirmed that the formed LS-Aß40-O-Apt complex attached onto the AuNP surfaces via interactions between LS-Aß40-O and Au, which led to electrostatic and steric stabilization of the AuNPs under high-salt conditions. On the basis of this outcome, a sensitive light-up nonthiolated aptasensor for LS-Aß40-O was achieved with a detection limit of 10.0 nM and a linear range from 35.0 to 700 nM in a 175 mM NaCl solution. Cerebrospinal-fluid (CSF) samples from healthy persons and Alzheimer's disease (AD) patients were successfully distinguished by using this proposed method. The concentrations of LS-Aß40-O in the CSF of AD patients were of nanomolar grade, but there was no detectable LS-Aß40-O in those of the healthy persons. This work provides a new insight into the interaction between Apt@AuNPs and Aß40-O and also develops a simple, rapid, highly selective and sensitive, and applicable method for LS-Aß40-O detection in real CSF samples, which is significant for the diagnosis of AD.

Interaction of divalent metal ions with human translocase of inner membrane of mitochondria Tim23.

The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. Tim23p, the core component of TIM23 complex, forms the import pore across the inner membrane and exerts a key function in the protein import. However, the interaction of divalent metal ions with Tim23p and the contribution in the interaction of presequence peptide with Tim23p are still unknown. Herein, we investigated the interaction of divalent metal ions with the intermembrane space domain of Tim23p (Tim23IMS) and the interaction of presequence peptides with Tim23IMS in presence of Ca(2+) ion by fluorescence spectroscopy in vitro. The static fluorescence quenching indicates the existence of strong binding between divalent metal ions and Tim23IMS. The order of the binding strength is Ca(2+), Mg(2+), Cu(2+), Mn(2+), and Co(2+) (from strong to weak). Moreover, the interaction of presequence peptides with Tim23IMS is weakened in presence of Ca(2+) ion, which implicates that Ca(2+) ion may play an important role in the protein import by TIM23 complex.

Resurfaced fluorescent protein as a sensing platform for label-free detection of copper(II) ion and acetylcholinesterase activity.

Protein engineering by resurfacing is an efficient approach to provide new molecular toolkits for biotechnology and bioanalytical chemistry. H39GFP is a new variant of green fluorescent protein (GFP) containing 39 histidine residues in the primary sequence that was developed by protein resurfacing. Herein, taking H39GFP as the signal reporter, a label-free fluorometric sensor for Cu(2+) sensing was developed based on the unique multivalent metal ion-binding property of H39GFP and fluorescence quenching effect of Cu(2+) by electron transfer. The high affinity of H39GFP with Cu(2+) (Kd, 16.2 nM) leads to rapid detection of Cu(2+) in 5 min with a low detection limit (50 nM). Using acetylthiocholine (ATCh) as the substrate, this H39GFP/Cu(2+) complex-based sensor was further applied for the turn-on fluorescence detection of acetylcholinesterase (AChE) activity. The assay was based on the reaction between Cu(2+) and thiocholine, the hydrolysis product of ATCh by AChE. The proposed sensor is highly sensitive (limit of detection (LOD) = 0.015 mU mL(-1)) and is feasible for screening inhibitors of AChE. Furthermore, the practicability of this method was demonstrated by the detection of pesticide residue (carbaryl) in real food samples. Hence, the successful applications of H39GFP in the detection of metal ion and enzyme activity present the prospect of resurfaced proteins as versatile biosensing platforms.

Interaction of presequence peptides with human translocase of inner membrane of mitochondria Tim23.

The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. Tim23p, the core component of TIM23 complex, forms the import pore across the inner membrane. However, the interaction between presequence peptides and Tim23p remains unclear. Herein, we investigated the interaction of presequence peptides with the intermembrane space domain of Tim23p (Tim23IMS) by fluorescence and micro-Raman spectroscopy. The fluorescence quenching revealed that the interaction between Tim23IMS and presequence peptides is mainly electrostatic interaction. Micro-Raman spectroscopy and ANS binding experiments showed that presequence peptides induce a more compact conformation of Tim23IMS. GST pull-down experiments and tryptophan fluorescence indicated that there is no interaction between Tim23IMS and Tim50IMS.

Interaction of divalent metal ions with human translocase of inner membrane of mitochondria Tim50.

The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. Tim50 is a major receptor for transporting the precursor protein across the mitochondrial inner membrane in TIM23 complex. However, the interaction of divalent metal ions with Tim50 and the contribution in the interaction of presequence peptide with Tim50 are still unknown. Herein, we investigated the interaction of divalent metal ions with the intermembrane space domain of Tim50 (Tim50(IMS)) and the interaction of presequence peptides with Tim50(IMS) in presence of Ca(2+) ion by fluorescence spectroscopy in vitro. The static fluorescence quenching indicates the existence of strong binding between divalent metal ions and Tim50(IMS). The order of the binding strength is Ca(2+), Mg(2+), Cu(2+), Mn(2+), and Co(2+) (from strong to weak). Moreover, the interaction of presequence peptides with Tim50(IMS) is weakened in presence of Ca(2+) ion, which implicates that Ca(2+) ion may play an important role in the protein import by TIM23 complex.

The structural characteristics of human preprotein translocase of the inner mitochondrial membrane Tim23: implications for its physiological activities.

The preprotein translocase of the inner mitochondrial membrane (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. Tim23 forms a pore for preprotein transportation in TIM23 complex, which spans the inner membrane with transmembrane segments and exposes a hydrophilic domain in the intermembrane space. In this study, we expressed and purified the intermembrane space (IMS) domain of human Tim23 (Tim23(IMS)). The far-UV CD spectra of Tim23(IMS) in native and denatured states revealed that the protein has a limited secondary structure and a not well-defined tertiary packing. Its Stokes radius was larger than both its expected size as a folded globular protein and the size determined by size exclusion chromatography. A large increase in 8-anilino-1-naphthalene-sulfonate (ANS) fluorescence (>50-fold) was observed, indicating that hydrophobic clusters are exposed at its surface. And GlobPlot/DisEMBL program predicted that the protein is in a loose folding state. We therefore conclude that, the non-bound hydrophilic domain of the human Tim23 is in a molten globule configuration with marginal stability. Furthermore, size exclusion chromatography and sedimentation equilibrium analysis showed that Tim23(IMS) exists as a dimer. And the results, showed by ANS binding and fluorescence quenching, indicated that a pH-dependent conformational change of Tim23(IMS) occurs, and at pH 4 and 3, it forms a compact structure.

Biomineralization synthesis of a near-infrared fluorescent nanoprobe for direct glucose sensing in whole blood.

A near-infrared (NIR) fluorescent nanoprobe that enables to circumvent the interference of background absorption and fluorescence in whole blood was developed for the direct sensing of blood glucose. Here, NIR fluorescent protein (iRFP) and glucose oxidase (GOx) were collectively deployed as the templates for the biomineralization of Mn2+ to prepare a NIR fluorescent nanoprobe (iRFP-GOx-MnO2 nanoparticles, iRGMs), in which the fluorescence of iRFP was effectively quenched by MnO2via energy transfer. When the iRGMs were mixed with whole blood samples, GOx can convert blood glucose into gluconic acid, as well as H2O2, which will reduce MnO2 and decompose the iRGMs. As a result, the NIR fluorescence of iRFPs was restored, providing a fluorometric assay for the direct detection of blood glucose. Owing to the high efficiency of the cascade reaction and the low background interference of the NIR fluorescence signal, accurate and rapid analysis of the glucose levels in whole blood samples was achieved using the iRGMs. Moreover, an iRGM-based paper device that only requires 5 microliters of samples was also demonstrated in the direct assay of blood glucose without any pretreatment, affording an alternative approach for the accurate monitoring of blood glucose levels.

Charge designable and tunable GFP as a target pH-responsive carrier for intracellular functional protein delivery and tracing.

A charge designable and tunable green fluorescent protein (GFP)-based protein delivery strategy was proposed. The acquired His29GFP selectively permeates the cell membrane at a target pH of 6.5 and escapes from the endosome efficiently. The delivered RNase A caused substantial mRNA degradation in HeLa cells, and proliferation inhibition in different cell lines and a 3D tumor model at pH 6.5.

Interaction of presequence with human translocase of the inner membrane of mitochondria Tim50.

The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. Tim50 is a major receptor for transporting the precursor protein across the mitochondrial inner membrane in the TIM23 complex. However, the interaction of prequence peptide with Tim50 is not well-known. Here, we investigated in vitro the interaction of presequence peptide with the intermembrane space domain of Tim50 (Tim50(IMS)) by micro-Raman and fluorescence spectra. The fluorescence quenching revealed that the interaction between Tim50(IMS) and presequence peptide is mainly electrostatic interaction, and the distances between Tim50(IMS) and presequence peptides are estimated by fluorescence resonance energy transfer. Micro-Raman spectra showed that presequence peptides induce a more compact conformation of Tim50(IMS), and synchronous fluorescence showed that the tyrosine or tryptophan fluorescence quenching molar ratio of presequence peptide to Tim50(IMS) is less than 3.