Rapid Equilibrated Colorimetric Detection of Protamine and Heparin: Recognition at the Nanoscale Liquid–Liquid Interface

Abstract

Demand for rapid quantitation of polyions such as heparin and protamine are ever growing. Previous paper-based and polymeric optical and electrochemical sensing devices required more than several hours for signal stabilization. Therefore, signals were acquired with fixed sample exposure time modes, which was time-consuming and technically demanding. We present here for the first time the optical detection of protamine and heparin in equilibrium mode with emulsified nanospheres. The method significantly shortens the response time from hours to typically less than 10 s and offers tunable, sensitive, and colorimetric detection within the clinically relevant range (10 to 100 mg/L) for heparin. The improved characteristics are attributable to the small size of the nanospheres (ca. 50 nm in diameter) as well as the reversible recognition at the nanoscale liquid−liquid interface. Detection of the anticoagulant heparin was also successfully demonstrated in human blood serum background.

Protamine/heparin optical nanosensors based on solvatochromism

“…For example, Xie and coworkers have reported that the signal of protamine sensors containing dinonylnaphthalenesulfonate (DNNS-), a commonly used protamine receptor, was found to completely saturate upon addition of serum to sensors, pointing to important matrix effects 25 and the need of extreme dilution of the serum samples. 26 In this work protamine and heparin were chosen as model target polyions as convention, and we describe protamineselective nanoscale optodes employing fundamentally novel sensing mechanism for assaying heparin in human plasma samples by protamine titration. The research first explores an ion exchange-based assay involving capillary coated sensing films, the basis of which was previously described for the detection of potassium in human serum.…”

“…For example, Xie and coworkers have reported that the signal of protamine sensors containing dinonylnaphthalenesulfonate (DNNS − ), a commonly used protamine receptor, was found to completely saturate upon addition of serum to sensors, pointing to important matrix effects 25 and the need for extreme dilution of serum samples. 26 …”

Recent improvements to the selectivity of extraction-based optical ion sensors

“…[54][55][56][57][58] Nanoparticle sensors were found to equilibrate within seconds. 34,53 The response time of membrane ISO sensors was recently improved by Hisamoto and coworkers by the use of very thin PVC membranes (ca. 140 nm).…”

“…Later, nanoparticle-based ISOs for polyion detection were introduced, 19,53 very recently resulting in highly sensitive and rapidly responding protamine sensors. The improvement of the response time was especially notable because the response time of the first cast film ISO sensors was very slow owing to long equilibration times (slow diffusion).…”

“…Unfortunately, polyion-selective nanoscale sensors were failing in undiluted serum or plasma, 34,53,59 requiring the use of diluted samples, while the membrane type sensors performed adequately in serum. 62 Likely the most significant difference from cast film sensors is their dramatically larger surface to volume ratio.…”