Estimating Poly(N-isopropylacrylamide) size in solution below the LCST using fluorescence correlation spectroscopy with non-covalent bound fluorophores

Abstract

Accurate size measurements of nanosized polymer chains in dilute solutions is important for understanding polymer behavior, however, these measurements can be challenging to implement accurately, and are technique dependent. Here we explore the use of Fluorescence Correlation Spectroscopy (FCS) to determine the size of single polymer chains in dilute solutions (<2 wt%). FCS, a technique with single molecule sensitivity, generally requires the use of covalently labelled polymers which can distort physicochemical behavior. Here, FCS based size measurements were based on the non-covalent interaction of fluorophores (Alexa 405, Atto 390, and Atto 425) with PNIPAm in water at 25 °C (below the Lower Critical Solution Temperature).FCS estimated size (hydrodynamic radius) of three different MW PNIPAm samples in water were: 5.0 ± 1.0 nm (28.5 kDa), 5.0 ± 1.0 nm (38 kDa), 3.3 ± 0.5 nm (55.5 kDa), in reasonable agreement with theoretical calculations. Accuracy was directly related to the fraction of PNIPAm-bound fluorophore, which were, for 1 wt% PNIPAm solutions in water: ∼11.5 % (Atto 390), ∼8.1 % (Atto 425), and 4 % (Alexa 405). This method has several advantages in that it does not require a covalent labelling of PNIPAm, it can be implemented on very small sample volumes, and allows for in-situ measurements.