Amplification-Free, High-Throughput Nanoplasmonic Quantification of Circulating MicroRNAs in Unprocessed Plasma Microsamples for Earlier Pancreatic Cancer Detection.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy that is often detected at an advanced stage. Earlier diagnosis of PDAC is key to reducing mortality. Circulating biomarkers such as microRNAs are gaining interest, but existing technologies require large sample volumes, amplification steps, extensive biofluid processing, lack sensitivity, and are low-throughput. Here, we present an advanced nanoplasmonic sensor for the highly sensitive, amplification-free detection and quantification of microRNAs (microRNA-10b, microRNA-let7a) from unprocessed plasma microsamples. The sensor construct utilizes uniquely designed -ssDNA receptors attached to gold triangular nanoprisms, which display unique localized surface plasmon resonance (LSPR) properties, in a multiwell plate format. The formation of -ssDNA/microRNA duplex controls the nanostructure-biomolecule interfacial electronic interactions to promote the charge transfer/exciton delocalization processes and enhance the LSPR responses to achieve attomolar (10-18 M) limit of detection (LOD) in human plasma. This improve LOD allows the fabrication of a high-throughput assay in a 384-well plate format. The performance of nanoplasmonic sensors for microRNA detection was further assessed by comparing with the qRT-PCR assay of 15 PDAC patient plasma samples that shows a positive correlation between these two assays with the Pearson correlation coefficient value >0.86. Evaluation of >170 clinical samples reveals that oncogenic microRNA-10b and tumor suppressor microRNA-let7a levels can individually differentiate PDAC from chronic pancreatitis and normal controls with >94% sensitivity and >94% specificity at a 95% confidence interval (CI). Furthermore, combining both oncogenic and tumor suppressor microRNA levels significantly improves differentiation of PDAC stages I and II versus III and IV with >91% and 87% sensitivity and specificity, respectively, in comparison to the sensitivity and specificity values for individual microRNAs. Moreover, we show that the level of microRNAs varies substantially in pre- and post-surgery PDAC patients (n = 75). Taken together, this ultrasensitive nanoplasmonic sensor with excellent sensitivity and specificity is capable of assaying multiple biomarkers simultaneously and may facilitate early detection of PDAC to improve patient care.

Photoswitchable Machine-Engineered Plasmonic Nanosystem with High Optical Response for Ultrasensitive Detection of microRNAs and Proteins Adaptively.

Modulating optoelectronic properties of inorganic nanostructures tethered with light-responsive molecular switches by their conformational change in the solid state is fundamentally important for advanced nanoscale-device fabrication, specifically in biosensing applications. Herein, we present an entirely new solid-state design approach employing the light-induced reversible conformational change of spiropyran (SP)-merocyanine (MC) covalently attached to gold triangular nanoprisms (Au TNPs) via alkylthiolate self-assembled monolayers to produce a large localized surface plasmon resonance response (∼24 nm). This shift is consistent with the increase in thickness of the local dielectric shell-surrounded TNPs and perhaps short-range dipole-dipole (permanent and induced) interactions between TNPs and the zwitterionic MC form. Water contact angle measurement and Raman spectroscopy characterization unequivocally prove the formation of a stable TNP-MC structural motif. Utilizing this form, we fabricated the first adaptable nanoplasmonic biosensor, which uses an identical structural motif for ultrasensitive, highly specific, and programmable detection of microRNAs and proteins at attomolar concentrations in standard human plasma and urine samples, and at femtomolar concentrations from bladder cancer patient plasma (n = 10) and urine (n = 10), respectively. Most importantly, the TNP-MC structural motif displays a strong binding affinity with receptor molecules (i.e., single-stranded DNA and antibody) producing a highly stable biosensor. Taken together, the TNP-MC structural motif represents a multifunctional super biosensor with the potential to expand clinical diagnostics through simplifying biosensor design and providing highly accurate disease diagnosis.

pH-Dependent Nitrotyrosine Formation in Ribonuclease A is Enhanced in the Presence of Polyethylene Glycol (PEG).

Protein nitration can occur as a result of peroxynitrite-mediated oxidative stress. Excess production of peroxynitrite (PN) within the cellular medium can cause oxidative damage to biomolecules. The in vitro nitration of Ribonuclease A (RNase A) results in nitrotyrosine (NT) formation with a strong dependence on the pH of the medium. In order to mimic the cellular environment in this study, PN-mediated RNase A nitration has been carried out in a crowded medium. The degree of nitration is higher at pH 7.4 (physiological pH) compared to pH 6.0 (tumor cell pH). The extent of nitration increases significantly when PN is added to RNase A in the presence of crowding agents PEG 400 and PEG 6000. PEG has been found to stabilize PN over a prolonged period, thereby increasing the degree of nitration. NT formation in RNase A also results in a significant loss in enzymatic activity.