Identification of Spherical and Nonspherical Proteins by a Solid-State Nanopore.

The three-dimensional structure of a protein plays an important role in protein dynamics in the biological system of human. By now, it remains a challenge to characterize and quantify the shape of a protein at the single-molecule level. The nanopores, as a novel single-molecule sensor, has been widely applied in many fields such as DNA sequencing and human diseases diagnosis. In this paper, we investigated the translocation of spherelike con.A and the prolate bovine serum albumin (BSA) under an electric field by a solid-state nanopore. By analyzing the ionic current, the con.A and the BSA could be characterized and differentiated due to their intrinsic shape difference. Because the prolate BSA will have the preferred orientations for a higher electric field, when it is residing inside the nanopore, multiple ionic current blockade levels will be observed. While for the spherical con.A, there is only one ionic current blockade level. The method presented here will be potentially applied to fingerprint a single protein as a new method having the features of low cost and high throughput in the near future.

Salt Gradient Improving Signal-to-Noise Ratio in Solid-State Nanopore.

As the single molecule detection tool, solid-state nanopores are being applied in more and more fields, such as medicine controlled delivery, ion conductance microscopes, nanosensors, and DNA sequencing. The critical information obtained from nanopores is the signal collected, which is the ionic block current caused by the molecules passing through the pores. However, the information collected is, in part, impeded by the relatively low signal-to-noise ratio of the current solid-state nanopore measurements. Here, we report that using a salt gradient across the nanopore could improve the signal-to-noise ratio when molecules translocate through Si3N4 nanopore. Furthermore, we demonstrate that the improved signal-to-noise ratio is connected with not only the value of surface charge but also that of a salt gradient between cis and trans sides of the nanopore.

An LC-MS/MS assay to determine plasma pharmacokinetics of cyclic thymic hexapeptide (cTP6) in rhesus monkeys.

A robust and simple method for absolute quantification of a novel bidirectional immunomodulatory drug candidate, cyclic thymic hexapeptide (cTP6), in rhesus monkey plasma was developed and validated by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Plasma proteins were precipitated by adding four volumes of acetonitrile. Peptides in the supernatant were separated by liquid chromatography on an Agilent Zorbax Eclipse Plus-C18 chromatographic column with gradient elution using 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in methanol (mobile phase B) at 0.2 mL/min. The analytes were identified by triple quadrupole mass spectrometry in positive ion-mode. The assay was linear over a concentration range of 10-5000 ng/mL for cTP6, with a lower limit of quantification (LLOQ) of 10 ng/mL. Intra- and inter-day precision of the assay at three concentrations were 1.51-7.70% with accuracy of 95.1-104.2%. The average recovery of cTP6 for three concentration levels was 59.6-64.0%. No significant matrix effect was observed. Peptide cTP6 was detected in plasma of live rhesus monkeys up to 6-8h after intra-muscular injection. The half-life was 2.24-2.95 h. The result revealed a nonlinear pharmacokinetic response to increasing doses of cTP6 (100, 200, 500 µg/kg). For the multiple dose study of cTP6, the drug did not accumulate during daily administration at 100 µg/kg for 7 consecutive days in rhesus monkeys.

Qualitative and quantitative studies on human B7.1-Fc fusion protein and the application in pharmacokinetic study in rhesus monkeys.

A sensitive, accurate, and precise enzyme immunoassay (EIA) for the quantification of intact human B7.1-Fc in rhesus monkey serum was validated, and the characteristics of B7.1 and Fc moiety of fusion protein were identified by surface plasmon resonance (SPR) and flow-cytometric method, respectively. B7.1-Fc bound to CD28 and CTLA-4 with K(d) values of 45.1 and 9.58 nM, respectively, which were very closed to the previous reports and the function of Fc moiety of fusion protein was also confirmed by Fc receptor binding assay and IL-8 releasing assay. To monitor the intact protein, the EIA method employed a sandwich scheme in which a multiclonal anti-human IgG (Fc specific) antibody and a monoclonal anti-human B7.1 antibody were served as capture and detection antibody, respectively. This EIA has a range of reliable response of 0.5-32 ng/ml. The LLOQ was established at 0.5 ng/ml. The intra-assay precision and accuracy were 6.1-8.8% and (3.0-9.0)%, respectively with the inter-assay precision and accuracy were 5.7-11.5% and (10.7-9.1)%, respectively. Stability was established under certain conditions and no significant differences were found. This validated EIA assay was then successfully employed in the assessment of pharmacokinetic behavior of B7.1-Fc in rhesus monkeys after intravenous infusion, and a non-linear characteristics was established across the investigated dosage range (32-320 µg/kg).