Therapeutic Small Interfering RNA Targeting Complement C3 in a Mouse Model of C3 Glomerulopathy.

Alternative pathway complement dysregulation with abnormal glomerular C3 deposits and glomerular damage is a key mechanism of pathology in C3 glomerulopathy (C3G). No disease-specific treatments are currently available for C3G. Therapeutics inhibiting complement are emerging as a potential strategy for the treatment of C3G. In this study, we investigated the effects of N-acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) targeting the C3 component of complement that inhibits liver C3 expression in the C3G model of mice with heterozygous deficiency of factor H (Cfh +/- mice). We showed a duration of action for GalNAc-conjugated C3 siRNA in reducing the liver C3 gene expression in Cfh +/- mice that were dosed s.c. once a month for up to 7 mo. C3 siRNA limited fluid-phase alternative pathway activation, reducing circulating C3 fragmentation and activation of factor B. Treatment with GalNAc-conjugated C3 siRNA reduced glomerular C3d deposits in Cfh +/- mice to levels similar to those of wild-type mice. Ultrastructural analysis further revealed the efficacy of the C3 siRNA in slowing the formation of mesangial and subendothelial electron-dense deposits. The present data indicate that RNA interference-mediated C3 silencing in the liver may be a relevant therapeutic strategy for treating patients with C3G associated with the haploinsufficiency of complement factor H.

Multiplexed Discrimination of Single Amino Acid Residues in Polypeptides in a Single SERS Hot Spot.

The SERS-based detection of protein sequences with single-residue sensitivity suffers from signal dominance of aromatic amino acid residues and backbones, impeding detection of non-aromatic amino acid residues. Herein, we trap a gold nanoparticle in a plasmonic nanohole to generate a single SERS hot spot for single-molecule detection of 2 similar polypeptides (vasopressin and oxytocin) and 10 distinct amino acids that constitute the 2 polypeptides. Significantly, both aromatic and non-aromatic amino acids are detected and discriminated at the single-molecule level either at individual amino acid molecules or within the polypeptide chains. Correlated with molecular dynamics simulations, our results suggest that the signal dominance due to large spatial occupancy of aromatic rings of the polypeptide sidechains on gold surfaces can be overcome by the high localization of the single hot spot. The superior spectral and spatial discriminative power of our approach can be applied to single-protein analysis, fingerprinting, and sequencing.

SERS discrimination of single DNA bases in single oligonucleotides by electro-plasmonic trapping.

Surface-enhanced Raman spectroscopy (SERS) sensing of DNA bases by plasmonic nanopores could pave a way to novel methods for DNA analyses and new generation single-molecule sequencing platforms. The SERS discrimination of single DNA bases depends critically on the time that a DNA strand resides within the plasmonic hot spot. In fact, DNA molecules flow through the nanopores so rapidly that the SERS signals collected are not sufficient for single-molecule analysis. Here, we report an approach to control the residence time of molecules in the hot spot by an electro-plasmonic trapping effect. By directly adsorbing molecules onto a gold nanoparticle and then trapping the single nanoparticle in a plasmonic nanohole up to several minutes, we demonstrate single-molecule SERS detection of all four DNA bases as well as discrimination of single nucleobases in a single oligonucleotide. Our method can be extended easily to label-free sensing of single-molecule amino acids and proteins.

Author Correction: Live Intracellular Biorthogonal Imaging by Surface Enhanced Raman Spectroscopy using Alkyne-Silver Nanoparticles Clusters.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Multiplex detection of urinary miRNA biomarkers by transmission surface plasmon resonance.

The clinical assessment of short-stranded nucleic acid biomarkers such as miRNAs could potentially provide useful information for monitoring disease progression, prompting definitive treatment decisions. In the past decade, advancements in biosensing technology have led to a shift towards rapid, real-time and label-free detection systems; as such, surface plasmon resonance (SPR) biosensor-based technology has become of high interest. Here, we developed an automated multiplex transmissive surface plasmon resonance (t-SPR) platform with the use of a capped gold nanoslit integrated microfluidic surface plasmon resonance (SPR) biosensor. The automated platform was custom designed to allow the analysis of spectral measurements using wavelength shift (dλ), intensity (dI) and novel area change (dA) for surface binding reactions. A simple and compact nanostructure based biosensor was fabricated with multiplex real-time detection capabilities. The sensitivity and specificity of the microfluidic device was demonstrated through the use of functionalised AuNPs for target molecule isolation and signal enhancement in combination with probes on the CG nanoslit surface. Our work allows for the multiplex detection of miRNA at femtomolar concentrations in complex media such as urine.

Live Intracellular Biorthogonal Imaging by Surface Enhanced Raman Spectroscopy using Alkyne-Silver Nanoparticles Clusters.

Live intracellular imaging is a valuable tool in modern diagnostics and pharmacology. Surface Enhanced Raman Spectroscopy (SERS) stands out as a non-destructive and multiplexed technique, but intracellular SERS imaging still suffers from interfering background from endogenous components. Here we show the assembly of small colloidal SERS probes with Raman signal in the cell-silent window of 1800-2900 cm-1 for biorthogonal intracellular SERS imaging of dopamine that was undistinguishable from the endogenous cell background. By linking colloidal silver nanoparticles with alkyne-dopamine adducts, clusters are formed by 2-6 nanoparticles spaced by tight interparticle gaps that exhibited high electric field enhancement and strong SERS signals of alkyne and dopamines. Due to the cell-silent signals of the alkyne, intracellular in-vitro Raman imaging shows that the dopamines on the internalized clusters remain distinguishable across the cytoplasm with good spatial resolution. Our method can be a general-purpose method for real-time imaging of biomolecules, such as proteins, peptides, DNA and drugs.

Urinary micro-RNA biomarker detection using capped gold nanoslit SPR in a microfluidic chip.

Successful diagnosis and treatment of many diseases depends on the availability of sensitive, reliable and low cost tools for the detection of the biomarkers associated with the diseases. Simple methods that use non-invasive biological samples are especially suitable for the deployment in the clinical environment. In this paper we demonstrate the application of a method that employs a capped gold nanoslit surface plasmon resonance (SPR) sensor and a microfluidic chip for the detection of a urinary nucleic acid biomarker in clinical samples. This method detects low concentrations of the biomarker in a relatively large volume (∼1 mL) of the sample. The method utilizes magnetic nanoparticles (MNPs) for the isolation of target molecules and signal enhancement in conjunction with surface plasmon resonance (SPR) on capped gold nanoslits. The ability of the method to detect urinary miRNA-16-5p in AKI patients was tested and the result was compared with the data obtained with the polymerase chain reaction (PCR). miRNA-16-5p has been found to be a specific and noninvasive biomarker for acute kidney injury (AKI). Our method allows the detection of the biomarker in the urine of AKI patients without amplification and labeling of the target molecules.

Label-free detection of rare cell in human blood using gold nano slit surface plasmon resonance.

Label-free detection of rare cells in biological samples is an important and highly demanded task for clinical applications and various fields of research, such as detection of circulating tumor cells for cancer therapy and stem cells studies. Surface Plasmon Resonance (SPR) as a label-free method is a promising technology for detection of rare cells for diagnosis or research applications. Short detection depth of SPR (400 nm) provides a sensitive method with minimum interference of non-targets in the biological samples. In this work, we developed a novel microfluidic chip integrated with gold nanoslit SPR platform for highly efficient immunomagnetic capturing and detection of rare cells in human blood. Our method offers simple yet efficient detection of target cells with high purity. The approach for detection consists of two steps. Target cells are firs captured on functionalized magnetic nanoparticles (MNPs) with specific antibody I. The suspension containing the captured cells (MNPs-cells) is then introduced into a microfluidic chip integrated with a gold nanoslit film. MNPs-cells bind with the second specific antibody immobilized on the surface of the gold nanoslit and are therefore captured on the sensor active area. The cell binding on the gold nanoslit was monitored by the wavelength shift of the SPR spectrum generated by the gold nanoslits.

Magnetic nanoparticle-enhanced SPR on gold nanoslits for ultra-sensitive, label-free detection of nucleic acid biomarkers.

We have demonstrated a detection method for the ultra-sensitive detection of an mRNA biomarker. The method utilizes functionalized magnetic nanoparticles (MNPs) for signal enhancement in conjunction with surface plasmon resonance (SPR) on gold nanoslits. The approach for detection includes double hybridization at two different specific locations in two steps. First, the biomarker target molecule is captured with MNPs, and second, MNPs carrying the target molecule are introduced to the SPR chip to hybridize with probes immobilized on the gold nanoslits. In this work, MNPs were applied for a dual purpose: to isolate the target molecule from the sample matrix to prevent non-specific binding and to enhance the SPR response. Gold nanoslits that provide SPR sensing were fabricated by nanoimprinting lithography on polycarbonate (PC) film. The film was integrated with a microliter volume microfluidic chip to form the SPR detection chip. This detection method was used to detect mRNA heterogeneous nuclear ribonucleoproteins (hnRNP B1) in two cancer cell lines, CL1-0 and CL1-5. hnRNP B1 is an mRNA biomarker that is overexpressed in lung cancer tissue in the early stage of cancer and can be found in the serum and plasma of lung cancer patients. A synthetic target molecule and extracted total RNA from the cell lines were used as samples. Without amplification and labeling of the target molecule, the SPR results demonstrate a specific and sensitive method for the detection of hnRNP B1 mRNA in extracted RNA from the two selected cell lines. The method is capable of measuring down to 30 fM of the target molecule in a 7 µl sample (corresponding to 1.26 × 10(5) molecules) without amplification and labeling of the target molecule.

The ratio of cervical fluid and serum human chorionic gonadotropin as a predictor of abortion.

BACKGROUND: Progressions in science and technology have generated several methods for delaying preterm delivery and abortion; therefore, discovering an easy, non-invasive, practical, and non-expensive predictive factor can help us to perform preventive methods in healthy pregnant women, without any risk factors. OBJECTIVE: To indicate an appropriate index for predicting abortion in early pregnancy. MATERIALS AND METHODS: In a prospective study, 73 pregnant women who had a singleton pregnancy, had no complications or history of abortion or disease, and were referred to Mahdieh and Taleghani Hospitals between 2007-2009, were evaluated. Blood and cervical fluid samples were obtained thrice from all patients: at the first visit, after 1 week, and 1-2 weeks later. They were followed up until the 12(th) week of gestation. RESULTS: Using the receiver-operator characteristic (ROC) curve analysis, 1.62 was obtained as the cut-off point for the cervical fluid: serum human chorionic gonadotropin concentration ratio; 14 patients (19.2%) experienced abortion, and 12 women (70.6%) had a ratio ≥1.62. Of the pregnant women with a ratio of <1.62, 3.6% had an abortion. CONCLUSION: Pregnant women who do not show any signs of abortion and have a high cervical fluid: serum HCG concentration ratio are at risk of abortion; therefore, the cut-off point might be an appropriate index for predicting abortion in early pregnancy.

A (13)C solid-state NMR analysis of vitamin D compounds.

(13)C cross-polarization/magic-angle spinning (CP/MAS) solid-state NMR spectroscopy has been employed to analyze four vitamin D compounds, namely vitamin D3 (D3), vitamin D2 (D2), and the precursors ergosterol (Erg) and 7-dehydrocholesterol (7DHC). The (13)C NMR spectrum of D3 displays a doublet pattern for each of the carbon atoms, while that of Erg contains both singlet and doublet patterns. In the cases of 7DHC and D2, the (13)C spectra display various multiplet patterns, viz. singlets, doublets, triplets, and quartets. To overcome the signal overlap between the (13)C resonances of protonated and unprotonated carbons, we have subjected these vitamin D compounds to 1D (1)H-filtered (13)C CP/MAS and {(1)H}/(13)C heteronuclear correlation (Hetcor) NMR experiments. As a result, assisted by solution NMR data, all of the (13)C resonances have been successfully assigned to the respective carbon atoms of these vitamin D compounds. The (13)C multiplets are interpreted due to the presence of s-cis and s-trans configurations in the alpha- and beta-molecular conformers, consistent with computer molecular modeling determined by molecular dynamics and energy minimization calculations. To further characterize the ring conformations in D3, we have successfully extracted chemical shift tensor elements for the (13)C doublets. It is demonstrated that (13)C solid-state NMR spectroscopy provides a robust and high sensitive means of characterizing molecular conformations in vitamin D compounds.