Ultrasensitive Detection of C-Reactive Protein by a Novel Nanoplasmonic Immunoturbidimetry Assay.

Nanotechnology has attracted much attention, and may become the key to a whole new world in the fields of food, agriculture, building materials, machinery, medicine, and electrical engineering, because of its unique physical and chemical properties, including high surface area and outstanding electrical and optical properties. The bottom-up approach in nanofabrication involves the growth of particles, and we were inspired to propose a novel nanoplasmonic method to detect the formation of nanoparticles in real time. This innovative idea may contribute to the promotion of nanotechnology development. An increase in nanometer particle size leads to optical extinction or density (OD)-value changes in our nanosensor chip at a specific wavelength measured in a generic microplate reader. Moreover, in applying this method, an ultrasensitive nanoplasmonic immunoturbidimetry assay (NanoPITA) was carried out for the high-throughput quantification of hypersensitive C-reactive protein (CRP), a well-known biomarker of cardiovascular, inflammatory, and tumor diseases. The one-step detection of the CRP concentration was completed in 10 min with high fidelity, using the endpoint analysis method. The new NanoPITA method not only produced a linear range from 1 ng/mL to 500 ng/mL CRP with the detection limit reduced to 0.54 ng/mL, which was an improvement of over 1000 times, with respect to regular immunoturbidity measurement, but was also effective in blood detection. This attractive method, combined with surface plasmon resonance and immunoturbidimetry, may become a new technology platform in the application of biological detection.

Novel nanostructure-coupled biosensor platform for one-step high-throughput quantification of serum neutralizing antibody after COVID-19 vaccination.

COVID-19 vaccination efficacy depends on serum levels of the neutralizing antibodies (NAs) specific to the receptor-binding domain of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Therefore, a high-throughput rapid assay capable of measuring the total SARS-CoV-2 NA level is urgently needed for COVID-19 serodiagnosis, convalescent plasma therapy, vaccine development, and assessment. Here, we developed a novel nanoplasmonic immunosorbent assay (NanoPISA) platform for one-step rapid quantification of SARS-CoV-2 NAs in clinical serum samples for high-throughput evaluation of COVID-19 vaccine effectiveness. The NanoPISA platform enhanced by the use of nanoporous hollow gold nanoparticle coupling was able to detect SARS-CoV-2 NAs with a limit of detection of 0.2 pM within 15 min without washing steps. The one-step NanoPISA for SARS-CoV-2 NA detection in clinical specimens yielded good results, comparable with those obtained in the gold-standard seroneutralization test and the surrogate virus-neutralizing enzyme-linked immunosorbent assay. Collectively, the one-step NanoPISA might be a rapid and high-throughput NA-quantification platform for evaluating the effectiveness of COVID-19 vaccines.

Heterogeneous nuclear ribonucleoprotein L facilitates recruitment of 53BP1 and BRCA1 at the DNA break sites induced by oxaliplatin in colorectal cancer.

Although oxaliplatin is an effective chemotherapeutic drug for treatment of colorectal cancer (CRC), tumor cells can develop mechanisms to evade oxaliplatin-induced cell death and show high tolerance and acquired resistance to this drug. Heterogeneous nuclear ribonucleoprotein L (hnRNP L) has been proved to play a critical role in DNA repair during IgH class switch recombination (CSR) in B lymphocytes, while, its role in CRC and chemotherapeutic resistance remain unknown. Our study aims to uncover an unidentified mechanism of regulating DNA double-strand breaks (DSBs) by hnRNP L in CRC cells treated by oxaliplatin. In present study, we observed that knockdown of hnRNP L enhanced the level of DNA breakage and sensitivity of CRC cells to oxaliplatin. The expression of key DNA repair factors (BRCA1, 53BP1, and ATM) was unaffected by hnRNP L knockdown, thereby excluding the likelihood of hnRNP L mediation via mRNA regulation. Moreover, we observed that phosphorylation level of ATM changed oppositely to 53BP1 and BRCA1 in the CRC cells (SW620 and HCT116) which exhibit synergistic effect by oxaliplatin plus hnRNP L impairment. And similar phenomenon was observed in the foci formation of these critical repair factors. We also found that hnRNP L binds directly with these DNA repair factors through its RNA-recognition motifs (RRMs). Analysis of cell death indicated that the RRMs of hnRNP L are required for cell survival under incubation with oxaliplatin. In conclusion, hnRNP L is critical for the recruitment of the DNA repair factors in oxaliplatin-induced DSBs. Targeting hnRNP L is a promising new clinical approach that could enhance the effectiveness of current chemotherapeutic treatment in patients with resistance to oxaliplatin.

Protein binding kinetics quantification via coupled plasmonic-photonic resonance nanosensors in generic microplate reader.

Almost no analytical assays, either colorimetric or fluorescence assays, for generic microplate readers is capable of dynamic measurements of protein-protein binding or the quantification of kinetic association and dissociation constants of protein interactions. On the other hand, protein binding kinetics quantification can be uniquely done on special expensive surface plasmon resonance (SPR) sensing equipment. Here we report the integration of coupled plasmonic-photonic resonance nanosensors in standard 96-well plate format and by using which, for the very first time, the demonstration of label-free dynamic SPR-like protein binding measurement and kinetics quantification in a generic microplate reader. Our low-cost label-free nanosensor plate enables very sensitive detection of immobilized protein interactions based on the transmission optical density (OD) value changes at specific wavelengths measured in a generic microplate reader. The relative end-point OD value changes show a good linear response with protein concentrations (from 0.05 to 50 µg/ml). And the protein quantification in serum results are consistent with the concurrent hospital lab tests. Most importantly, the kinetic association and dissociation constants of protein interactions in our sensor plate wells are determined by time-lapse dynamic OD value measurement in the generic microplate reader. Enabled by our unique nanosensor plate, SPR-like measurement of protein binding kinetics is now available using generic microplate reader ubiquitous in many chemistry and biomedical research labs.

C-Reaction Protein Detection in Human Saliva by Nanoplasmonic Color Imaging.

A label-free and highly sensitive imaging sensor based on plasmonic-photonic interaction in a gold-titanium dioxidegold metal-insulator-metal (MIM) plasmonic nanocup array is reported. The sensor can detect proteins and exhibits superior performance in visible light sensing. This device enables very sensitive detection of an increase in superstrate refractive index based on changes in the red channel intensity of color imaging. Compared to other conventional plasmonic sensors, our device achieves transmission imaging detection by using normal white light and minimizes instrumentation requirement. In this study, we used the device to detect C-reaction protein (CRP) level in saliva, which is widely tested to help make clinical decisions in different diseases and disorders. The intensity imaging showed a good linear response between CRP concentration (from 5 to 100 ng/mL) and relative intensity change in the device. The lowest concentration of CRP that could be detected was 5 ng/mL. Moreover, it could achieve a positive detection in saliva from patients when the CRP level in serum was 3.2 µg/mL. Owing to the high performance of the MIM plasmonic nanocup array, the proposed device is promising for future portable optical sensing with visible light illumination and imaging.