Mass Transfer from Ion-Sensing Component-Loaded Nanoemulsions into Ion-Selective Membranes: An Electrochemical Quartz Crystal Microbalance and Thin-Film Coulometry Study.

Recent work has shown that ion-selective components may be transferred from nanoemulsions (NEs) to endow polymeric membranes with ion-selective sensing properties. This approach has also been used for nanopipette electrodes to achieve single-entity electrochemistry, thereby sensing the ion-selective response of single adhered nanospheres. To this date, however, the mechanism and rate of component transfer remain unclear. We study here the transfer of lipophilic ionic compounds from nanoemulsions into thin plasticized poly(vinyl chloride) (PVC-DOS) films by chronoamperometry and quartz crystal microbalance. Thin-film cyclic coulovoltammetry measurements serve to quantify the uptake of lipophilic species into blank PVC-DOS membranes. Electrochemical quartz crystal microbalance data indicate that the transfer of the emulsion components is insignificant, ruling out simple coalescence with the membrane film. Ionophores and ion-exchangers are shown to transfer into the membrane at rates that correlate with their lipophilicity if mass transport is not rate-limiting, which is the case with more lipophilic compounds (calcium and sodium ionophores). On the other hand, with less lipophilic compounds (valinomycin and cation-exchanger salts), transfer rates are limited by mass transport. This is confirmed with rotating disk electrode experiments in which a linear relationship between the diffusion layer thickness and current is observed. The data suggests that once the nanoemulsion container approaches the membrane surface, transfer of components occur by a three-phase partition mechanism where the aqueous phase serves as a kinetic barrier. The results help better understand and quantify the interaction between nanoemulsions and ion-selective membranes and predict membrane doping rates for a range of components.

Optical Detection of Heparin in Whole Blood Samples Using Nanosensors Embedded in an Agarose Hydrogel.

Point-of-care quantification of the anticoagulant heparin still remains a significant clinical challenge as the reference method (colorimetric anti-factor Xa assay) cannot be performed in whole blood. Our group recently put forth the novel optical nanosensing principle using an ionic solvatochromic dye as a signal transducer. These nanosensors demonstrated significantly improved selectivity and sensitivity compared to ion-exchange-type polyion nanosensors and enabled protamine/heparin quantification in blood plasma samples. However, because the readout is absorbance-based, they are still not suitable for whole blood measurements. To overcome the background absorbance of blood, the nanosensors were here embedded in an agarose hydrogel capable of filtering out red blood cells while allowing plasma components to diffuse into the gel. Calibration curves for both protamine and heparin were successfully obtained in buffer, undiluted plasma, and undiluted whole blood using different colorimetric image analysis methods and a simple experimental setup.

Response Mechanism of Hyperpolarization-Based Polyion Nanosensors.

The last decade has witnessed a rapid development of nano- and microparticle-based optical ion sensors, including ion-selective optodes (ISOs). While the application of nano-ISOs has shown promising performance for sensing inorganic ions, polyion sensing using nanoscale ISOs has encountered significant interference in complex samples such as blood plasma. Recently, we have reported on a new polyion sensing principle that operates through a novel mechanism to overcome this challenge. The new sensing mechanism showed improved characteristics not observed with conventional ion-exchange type sensors, but the precise mechanism of operation remained thus far unclear. This paper aims to clarify how protamine, the arginine-rich target polycation, behaves during optical signal transduction to give dramatically improved selectivity. Based on thermodynamic data, sensor performance and ζ-potential analysis, two discrete phases of protamine extraction are identified. Initially, protamine extracts into the bulk nanosensor phase, a process that is concurrent with the optical signal change. This is then followed by protamine accumulation onto the nanosensor surface, which starts only upon saturation of the optical signal change. The data indicate that the improved selectivity is due to the inability of small ions to form a sufficiently strong interaction with an active sensing ingredient, DNNS-. Any exchange of one inorganic cation for another therefore remains optically silent, suppressing matrix effects. Moreover, the recognition of protamine is shown to be an exhaustive extraction process, making the response independent of the nature and concentration of the initial small cation in the nanosensor phase.

Chemo- and Regioselective Multiple C(sp2 )-H Insertions of Malonate Metal Carbenes for Late-Stage Functionalizations of Azahelicenes.

Chiral quinacridines react up to four times, step-by-step, with α-diazomalonates under RuII and RhII catalysis. By selecting the catalyst, [CpRu(CH3 CN)3 ][PF6 ] (Cp=cyclopentadienyl) or Rh2 (oct)4 , chemo and regioselective insertions of derived metal carbenes are achieved in favor of mono- or bis-functionalized malonate derivatives, respectively, (r.r.>49 : 1, up to 77 % yield, 12 examples). This multi-introduction of malonate groups is particularly useful to tune optical and chemical properties such as absorption, emission or Brønsted acidity but also cellular bioimaging. Density-functional theory further elucidates the origin of the carbene insertion selectivity and also showcases the importance of conformations in the optical response.

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

Optical sensors continue to demonstrate tremendous potential across a wide range of applications due to their high versatility and low cost. This feature article will focus on a number of recent advances made in improving the performance of extraction-based optical ion sensors within our group. This includes the progress of anchored solvatochromic transduction to provide pH and sample volume independent optical responses in nanoemulsion-based sensors. A recent breakthough is in polyion sensing in biological fluids that uses a novel indirect transduction mechanism that significantly improves the selectivity of dinonylnaphthalenesulfonate-based protamine sensors and its potential applications beyond polyion sensing. The role of particle stabilizers in relation to the response of emulsified sensors is shown to be important. Current challenges in the field and possible opportunities are also discussed.

Hyperpolarized Solvatochromic Nanosensors towards Heparin Sensing in Blood.

Heparin quantification at the point of care has been of medical interest for years but a suitable point of care measurement method for whole blood is still elusive. Our group has recently developed a nanoparticle-based optical sensor for protamine that allows for heparin quantification in plasma. This work discusses the effect of the transducing-dye structure and the promise of embedding the sensors in an agarose gel for avoiding red blood cell interference.

Colorimetric ratiometry with ion optodes for spatially resolved concentration analysis.

The deprotonation degree of the lipophilic pH indicator dye (chromoionophore) in ionophore-based ion optodes (so-called bulk optodes) has traditionally been measured spectrophotometrically. This makes it difficult to obtain spatially resolved concentration information, for example in the study of heterogenous systems. This article reports on a new colorimetric method that relies on a ratiometric image analysis. The acquision of image data allows one to map the deprotonation degree in two dimensions, which in turn is used to obtain the spatially-resolved ion concentration of the image. Using the detection of potassium as an example, the deprotonation degree data calculated on the basis of image analysis correlate quantitatively with those from spectrophotometry. They showed no dependence on the type of camera used in spite of their different gamma correction values and spectral sensitivities, as expected from theory. As an example, the method is successfully applied to the pixel level analysis of an ensemble of pictures acquired at different times to spatially and temporally observe potassium ion diffusion into an agarose gel containing a potassium-selective optical sensor microemulsion.

Protamine/heparin optical nanosensors based on solvatochromism.

Optical nanosensors for the detection of polyions, including protamine and heparin, have to date relied upon ion-exchange reactions involving an analyte and an optical transducer. Unfortunately, due to the limited selectivity of the available ionophores for polyions, this mechanism has suffered from severe interference in complex sample matrices. To date no optical polyion nanosensors have demonstrated acceptable performance in serum, plasma or blood. Herein we describe a new type of nanosensor based on our discovery of a "hyper-polarizing lipophilic phase" in which dinonylnaphthalenesulfonate (DNNS-) polarizes a solvatochromic dye much more than even an aqueous environment. We have found that the apparent polarity of the organic phase is only modulated when DNNS- binds to large polyions such as protamine, unlike singly charged ions that lack the cooperative binding required to cause a significant shift in the distribution of the polarizing DNNS- ions. Our new sensing mechanism allows solvatochromic signal transduction without the transducer undergoing ion exchange. The result is significantly improved sensitivity and selectivity, enabling for the first time the quantification of protamine and heparin in human plasma using optical nanosensors that correlates with the current gold standard analysis method, the anti-Xa factor assay.

Emulsion Doping of Ionophores and Ion-Exchangers into Ion-Selective Electrode Membranes.

Ion-selective electrodes (ISEs) are widely used analytical devices to selectively measure ionic species. Despite significant advances in recent years, ion-selective membranes are still mostly prepared in the same manner, by preloading the selective components into a solvent that is subsequently cast into a membrane or film. This paper describes an alternative method to prepare ISE membranes by mass transfer of the sensing components from an emulsion phase. Specifically, blank (undoped) plasticized poly(vinyl chloride) (PVC) membranes mounted into an electrode body are immersed into an aqueous solution containing analyte ions and an appropriate emulsion of the desired sensing components to allow their transfer into the membrane. The concept is demonstrated with conventional membrane electrodes containing an inner solution as well as all-solid-state electrodes. It is shown to be universally useful for the realization of ISEs for K+, Na+, Ca2+, and NO3-.

Optical Sensing with a Potentiometric Sensing Array by Prussian Blue Film Integrated Closed Bipolar Electrodes.

The simultaneous optical readout of a potentiometric sensor array of ion-selective electrodes (ISEs) based on PVC membranes is described here for the first time. The optical array consists of electrochromic Prussian Blue (PB) films in multiple closed ion-selective bipolar electrodes (BPEs), which gives a physical separation between the optical detection and sample compartments. The potential-dependent turnover of PB generates Prussian White (PW). A near-Nernstian response of the PB film is confirmed by colorimetric absorbance experiments as a function of applied potential. In the combined bipolar electrode cell, the overall potential is kept constant with a single potentiostat over the entire array where each PB spot indicates the potential change of an individual connected potentiometric probe. For cation-selective electrodes, the absorbance or blue intensity of the connected PB film is enhanced with increasing target cation activity. The colorimetric absorbance changes are simultaneously followed by a digital camera and analyzed by Mathematica software. A multiple cation-BPE array allows one to achieve simultaneous quantitative analysis of potassium, sodium, and calcium ions, demonstrated here in highly colored fruit juices. Mass transport at the PB thin film is shown not to be rate-limiting. The measuring ranges can be tuned in a wide range by potential control. The PB film exhibits greatly improved reproducibility and stability as compared to previous work with a ferroin redox probe confined in a thin solution layer.

Colorimetric absorbance mapping and quantitation on paper-based analytical devices.

A wide range of microfluidic paper-based analytical devices (µPADs) have been developed in the last decade. Despite this, the quality of colorimetric analysis has not substantially improved as the data is vulnerable to heterogeneous color distribution (e.g., coffee ring effects), non-uniform shapes of colored detection area, and noise from the underlying paper structure. These limitations are here addressed by a colorimetric method to quantify freely discharged dye on paper substrate, without the need for a defined channel or hydrophobic barrier. For accurate quantification, colorimetric absorbance values are calculated for each pixel based on the recorded RGB values and noise from the paper structure eliminated, to extract accurate absorbance information at the pixel level. Total analyte quantity is then calculated through the conversion of absorbance values into quantity values for each pixel followed by integration across the entire image. The resulting quantity is shown to be independent of the shape of the applied colored dye spot, with a cross, circle or rod shape all giving the same quantity information. The approach is applied to a capillary-based potassium-selective sensor, where the sample solution is loaded with the dye thioflavin T (ThT) obtained by quantitative exchange with K+ in a sensing capillary, which is discharged onto a bare paper substrate without any channels. The resulting dye quantity is successfully obtained by flatbed scanner and smartphone. The successful automated computation of colorimetric data on µPADs will help realize simpler paper-based assay and reaction systems that should be more applicable to addressing real world analytical problems.

Ultrasensitive Seawater pH Measurement by Capacitive Readout of Potentiometric Sensors.

Potentiometric pH probes remain the gold standard for the detection of pH but are not sufficiently sensitive to reliably detect ocean acidification at adequate frequency. Here, potentiometric probes are made dramatically more sensitive by placing a capacitive electronic component in series to the pH probe while imposing a constant potential over the measurement circuit. Each sample change now triggers a capacitive current transient that is easily identified between the two equilibrium states, and is integrated to reveal the accumulated charge. This affords dramatically higher precision than with traditional potentiometric probes. pH changes down to 0.001 pH units are easily distinguished in buffer and seawater samples, at a precision (standard deviation) of 28 µpH and 67 µpH, respectively, orders of magnitude better than what is possible with potentiometric pH probes.

Quantification of Colorimetric Data for Paper-Based Analytical Devices.

Colorimetric measurements by image analysis, giving RGB or HSV data, have become commonplace with optical indicator-based assays and as a readout for paper-based analytical devices (PADs). Yet, most works on PADs tend to ignore the quantitative relationship between color data and concentration, which may hamper their establishment as analytical devices and make it difficult to properly understand chemical or biological reactions on the paper substrate. This Perspective Article discusses how image color data are computed into colorimetric absorbance values that correlate linearly to dye concentration and compare well to traditional spectrophotometry. Thioflavin T (ThT), Neutral Red (NR), and Orange IV are used here as model systems. Absorbance measurements in solution correlate well to image data (and Beer's law) from the color channel of relevance if the gamma correction normally used to render the picture more natural to the human eye is removed. This approach also allows one to correct for color cast and variable background color, which may otherwise limit quantitation in field measurements. Reflectance measurements on paper color spots are equally found to correlate quantitatively between spectroscopy and imaging devices. In this way, deviations from Beer's law are identified that are explained with dye interactions on the paper substrate.

Equipment-Free Detection of K+ on Microfluidic Paper-Based Analytical Devices Based on Exhaustive Replacement with Ionic Dye in Ion-selective Capillary Sensors.

A distance-based analysis of potassium ion (K+) is introduced that is performed on a microfluidic paper-based analytical device (µPAD) coupled to an ion-selective capillary sensor. The concept is based on two sequential steps, the selective replacement of analyte ion with an ionic dye, and the detection of this dye in a distance-based readout on paper. To achieve the first step, the capillary sensor holds a poly(vinyl chloride) (PVC) membrane film layer plasticized by dioctyl sebacate (DOS) that contains the potassium ionophore valinomycin, a lipophilic cation-exchanger and the ionic indicator Thioflavin T (ThT) on its inner wall. Upon introduction of the sample, K+ in the aqueous sample solution is quantitatively extracted into the film membrane and replaced with ThT. To convert the ion exchange signal into a distance-based analysis, this solution was dropped onto the inlet area of a µPAD to flow the ThT along a channel defined by wax printing, resulting in the electrostatic binding of ThT to the cellulose carboxylic groups. The initial amount of K+ determines the amount of ThT in the aqueous solution after ion-exchange, and consequently the distance of ThT-colored area reflects the sample K+ concentration. The ion exchange reaction was operated in a so-called "exhaustive sensing mode" and gave a distinct response in a narrow range of K+ concentration (1-6 mM) that cannot be achieved by the classical optode sensing mode. The absence of hydrogen ions from the equilibrium competition of the capillary sensor contributed to a complete pH-independence, unlike conventional optodes that contain a pH sensitive indicator. A very high selectivity for K+ over Na+ and Ca2+ has been confirmed in separate solutions and mixed solutions tests. K+ measurements in pooled serum samples at concentrations between 2 and 6 mM are successfully demonstrated on a temperature controlled support.