Advanced Compositional Analysis of Nanoparticle-polymer Composites Using Direct Fluorescence Imaging

Summary

Here we present a reliable method to monitor the incorporation of nanoparticles into a polymer host matrix via swell encapsulation. We show that the surface concentration of cadmium selenide quantum dots can be accurately visualized through cross-sectional fluorescence imaging.

Abstract

The fabrication of polymer-nanoparticle composites is extremely important in the development of many functional materials. Identifying the precise composition of these materials is essential, especially in the design of surface catalysts, where the surface concentration of the active component determines the activity of the material. Antimicrobial materials which utilize nanoparticles are a particular focus of this technology. Recently swell encapsulation has emerged as a technique for inserting antimicrobial nanoparticles into a host polymer matrix. Swell encapsulation provides the advantage of localizing the incorporation to the external surfaces of materials, which act as the active sites of these materials. However, quantification of this nanoparticle uptake is challenging. Previous studies explore the link between antimicrobial activity and surface concentration of the active component, but this is not directly visualized. Here we show a reliable method to monitor the incorporation of nanoparticles into a polymer host matrix via swell encapsulation. We show that the surface concentration of CdSe/ZnS nanoparticles can be accurately visualized through cross-sectional fluorescence imaging. Using this method, we can quantify the uptake of nanoparticles via swell encapsulation and measure the surface concentration of encapsulated particles, which is key in optimizing the activity of functional materials.

Introduction

The application of nanomaterials has long served as an area of increasing interest for novel technologies.^1-3 This has included the growing use of nanoparticles in everyday items, including cosmetics, clothes, packaging and electronics.^4-6 A major drive toward using nanoparticles in functional materials stems from their higher reactivity relative to the materials, in addition to the ability to tune properties by variation in particle size.⁷ One further advantage is the capability to easily form composite materials, introducing crucial properties to the host matrix, such as catalytic functionality, material strengthening and tuning of electrical properties.^8-12

Nanoparticle-polymer composite materials can be achieved through a range of techniques, the simplest of which is direct integration of the desired nanoparticles during the fabrication of the host matrix.^13,14 This results in a homogenous material with an even spacing of nanoparticulate material throughout. However, many applications only require the active material to be present at the external interfaces of the nanocomposites. As a result, direct incorporation does not result in efficient use of sometimes costly nanoparticle material as there is much nanoparticle waste through the bulk of the material.^15,16 To achieve direct incorporation, the nanoparticles must also be compatible with host matrix formation. This may be challenging, especially in syntheses that require multifaceted reactions such as in the case of thermosetting polymers that are typically facilitated by metal complex catalysts mechanisms that may be affected by highly active nanoparticles.¹⁴

The considerable disadvantages associated with direct nanoparticle incorporation during the polymer synthesis, has led to the development of techniques aimed to limit nanoparticle incorporation to the surface layer.^17-21 Swell encapsulation is one of the most successful strategies reported in the literature, to achieve high surface nanoparticle concentrations, with limited wastage in the polymer bulk.^17-19 The technique utilizes the solvent driven swelling of polymer matrices, allowing for the incursion of molecular species and nanoparticles. Upon removal of the swelling solvent, the species within the matrix become fixed into place, with the highest concentration of species localized at the surface. To date, most of the reported uses of swell encapsulation are directed toward the fabrication of antimicrobial polymers, where it is key that the active agents are at the material surface. While many of these reports show enhanced antimicrobial activity, the precise surface nanoparticle composition is rarely probed in detail. Crick et al. recently demonstrated a method for the direct visualization of nanoparticle incursion, providing crucial insight into the kinetics and surface nanoparticle concentrations achieved by swell encapsulation.²²

This work details the synthesis of cadmium selenide quantum dots (QD), their swell encapsulation into polydimethylsiloxane (PDMS) and the direct visualization of their incorporation using fluorescence imaging. The effect of varying swell encapsulation time and nanoparticle concentration in the swelling solution is explored. The fluorescence visualization technique allows for the direct imaging of nanoparticle incursion into the PDMS and demonstrates that the highest concentration of QDs is at the material surface.

Protocol

1. Preparation of CdSe/ZnS Core/Shell Quantum Dots

1. Preparation of the trioctylphosphine (TOP)-Se solution
   1. Prepare a 0.5 M solution of selenium in TOP by mixing the appropriate amount of Se into TOP in a Schlenk flask under nitrogen or in a glovebox (8 ml required per reaction, typically 0.4 g dissolved in 10 ml of TOP).
   2. Stir the mixture to dissolve the Se for 1 hr, resulting in a grey solution of the TOP-Se complex.
   3. Ensure the solution is then freeze-pump-thaw degassed 5 times. The resulting stock solution can be stored under nitrogen for 3 months.
2. Preparation of the CdSe Cores
   1. Weigh out cadmium oxide (51 mg, 0.4 mmol), TOP oxide (3.7 g, 9.6 mmol), hexadecylamine (1.93 g, 8 mmol) and 1-dodecylphosphonic acid (0.22 g, 0.88 mmol) and combine into a three neck, 250 ml round bottom flask. Add a stir bar.
   2. Close two necks with septa and ensure the third is attached to a long reflux condenser and a nitrogen/vacuum Schlenk line. Insert a heating mantle temperature probe through one septum directly into the mixture. Pump/refill the flask with nitrogen five times.
   3. Heat the flask to 320 °C and stir the melt for 1 hr under a nitrogen atmosphere.
   4. Lower the temperature of the mantel to 270 °C and then use a large syringe and wide bore needle (20 ml, 3 mm bore) to degas with nitrogen 5 times.
   5. Take up 8 ml of the TOP-Se solution (step 1.1) and inject carefully but rapidly into the three neck flask, through the septum.
   6. Stir the reaction at 270 °C for between 30 sec and 10 min to control the size of particles produced. For red emission (~ 600 nm), 7-9 min is suitable.
   7. Prepare a bowl of boiling water (large enough to submerge half of the reaction vessel) and place next to the reaction mixture. After the reaction time is complete, rapidly cool the reaction in the boiling water with swirling.
      CAUTION: Cooling can cause the flask to crack. Take great care and wear thick impervious gloves.
   8. Once cool, inject 10 ml of chloroform into the flask to dissolve all the products, and divide the mixture between two 50 ml centrifuge tubes.
   9. Top up each tube up to 50 ml with EtOH and centrifuge at 3,600 × g for 10 min to precipitate the particles. Discard the supernatant and re-disperse the pellets in a total of 10 ml of n-hexane.
3. ZnS Shelling of the CdSe Core:
   1. Add the cores in hexane to a 100 ml round bottom flask containing zinc diethyldithiocarbamate (0.5 g, 1.4 mmol), oleylamine (3 ml, 9.12 mmol), trioctylphosphine (3 ml, 6.73 mmol) and 1-octadecene (10 ml). Add a stir bar. Exchange the reaction atmosphere to nitrogen.
   2. Heat the reaction on a hotplate-stirrer at 3.3 °C/min under partial vacuum until 70 °C, and remove the hexane using the Schlenk line.
   3. Switch the atmosphere to nitrogen and continue heating at this rate to 120 °C. Stir at 120 °C for 2 hr.
   4. Allow the reaction to cool, and split the mixture between 2 × 50 ml centrifuge tubes. Ensure the tubes are topped up to 50 ml with EtOH to precipitate the particles and centrifuge at 3,600 × g for 10 min.
   5. Discard the supernatant and re-disperse the pellets in a total of 10 ml of n-hexane.
   6. Centrifuge this solution (3,600 x g, 10 min) once more to remove any insoluble impurities, before being decanted into a sample tube, and store in the fridge (4 °C) under a nitrogen atmosphere for up to three months.

2. Swelling Encapsulation of Nanoparticles into PDMS

1. Swelling Solution Preparation:
   1. Prepare a stock solution of CdSe QDs by mixing 36 ml of n-hexane with 4 ml of CdSe QD dispersion (as synthesized) and stir the solution magnetically.
   2. Set aside two vials each containing 9 ml of the stock solution as designated swelling solutions.
   3. Use the remainder of the stock solution for the preparation of further swelling solutions of varying QD concentrations. Prepare three swelling solutions of decreasing QD concentration by diluting the stock solution to give a 66% (v/v) solution, 50% solution and 33% solution.
      1. Prepare the 66% (v/v) solution by mixing 6 ml of the CdSe QD stock solution with 3 ml n-hexane. Stir the solution magnetically to ensure complete mixing.
      2. Prepare the 50% (v/v) solution by mixing 4.5 ml of the CdSe QD stock solution with 4.5 ml n-hexane. Stir the solution magnetically to ensure complete mixing.
      3. Prepare the 33% (v/v) solution by mixing 3 ml of the CdSe QD stock solution with 6 ml n-hexane. Stir the solution magnetically to ensure complete mixing.
   4. Store all of the QD solutions under dark conditions, at room temperature.
2. Polymer Preparation and QD Incorporation - Varying the QD Concentration in the Swelling Solution:
   1. Cut out four medical grade silicone squares (11 mm x 11 mm) using a fresh scalpel blade.
   2. Immerse a medical grade silicone square in each of the four swelling solutions of varying % QD concentration: stock solution, 66% (v/v), 50% (v/v) and 33% (v/v). Allow the polymer samples to swell for 24 hr under dark conditions, and at room temperature.
   3. Remove the swollen polymer samples from the respective swelling solutions and air dry under dark conditions for 48 hr, during which time the residual solvent evaporates and the polymers shrink back to their initial dimensions.
   4. Wash the QD-incorporated samples thoroughly with deionized water to remove any surface bound materials.
3. Polymer Preparation and QD Incorporation - Varying the Polymer Exposure Time to the Swelling Solution:
   1. Prepare four more medical grade silicone squares (11 mm x 11 mm), as noted in 2.2.1.
   2. Immerse the medical grade silicone squares in the stock swelling solution for varying time periods: 1 hr, 3 hr, 6 hr and 24 hr.
   3. After removal from the swelling solution, air dry the swollen polymer samples under dark conditions for 48 hr, such that the sample shrinks back to its previous dimensions.
   4. Wash the QD-incorporated samples thoroughly with deionized water to remove any surface bound materials or residual solvent.

3. Visualization of Nanoparticle Swell Encapsulation into PDMS

1. Silicone Sample Preparation:
   1. Cut out two silicone squares with a fresh scalpel blade (5.5 mm x 11 mm). Ensure that this exposes the internal surface of the silicone samples.
2. Fluorescence Imaging:
   1. Place the silicone samples on a microscope slide for imaging, ensuring that the freshly cut side of the polymer makes full contact with the glass slide. Press the silicone portion down lightly to ensure a smooth contact with the microscope slide. Place the sample on the stage of the microscope.
   2. Complete lifetime fluorescence measurements using a 488 nm spectrally filtered laser line comprising of 5 psec pulses at a rate of 20 MHz or similar.²³ Use an acousto-optic tunable filter system, to directly couple to the laser output, in order to generate the 488 nm laser line. Focus the laser beam using a custom-built laser-scanning unit (see supplementary information), which is reflected by a dichroic mirror (488 nm) into the back aperture of a 10X objective and then onto the sample.
      1. Collect the fluorescence emission with the same objective, that then passes through the same dichroic mirror. Direct this light towards an avalanche photodiode operating in single photon counting mode. Process lifetime measurements using a time-correlated single photon counting (TCSPC) board.²³
   3. Record the instrument response function (IRF) at the beginning and the end of each experimental session.²³
      Note: The recorded signal in the TCSPC experiment should show the time delay between the photons arrival at the detector and the production of the subsequent laser pulse. This time delay however, should be convolved with the IRF of the measuring apparatus. Hence, the IRF is measured as the response of the instrument to 100 nM Auramine O, which has a significantly shorter lifetime (~100 psec) compared to the instrumental response.
   4. Extract the lifetime by non-linear fitting of the exponential decay of the fluorescent intensity and deconvolute this from the IRF using a maximum likelihood estimator (MLE) algorithm^25-27. The MLE (γ_j) is calculated as
      
      Where n_i is the number of photon counts in channel i, k is the number of channels (or bins) for each fluorescence decay, p_i(j) is the probability that a group of photons will fall in channel i if the particles have a lifetime j, and N is the total number of counts for a given decay.
   5. Record each sample fluorescence intensity and lifetime data for 5 min, creating a continuous scan consisting of 264 images with size 512 × 512 pixels. Combine these to provide two-dimensional fluorescence intensity, lifetime and intensity weighted lifetime maps, with a calculated MLE with threshold of 150 photons and process this using MATLAB.

Results

The quantum dots exhibited red fluorescence, with a lambda max of approximately 600 nm.^22,28 The red emission was due to the confinement of the exciton by the quantum rod whose size dimensions are within the strong confinement regime. Li et al. showed that for quantum rods, the emission shifts to lower energy with an increase in either width or length of the rod. They further showed that the emission mainly determined by the lateral confinement, which plays an importan...

Discussion

Cross-sectional fluorescence imaging allows for direct visualization of nanoparticles during swell encapsulation. The kinetics of encapsulation has been shown, with the drive toward a high nanoparticle surface concentration demonstrated. The extent of nanoparticle incorporation is shown to vary with swell encapsulation time (described in section 2.3), with the total amount of incorporated nanoparticles increasing as this time is extended, with the particle concentration localized at the surface if the polymer samples are...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Polydimethylsiloxane sheets	NuSil	-	Medical Grade
Oleylamine	Sigma Aldrich	O7805	Technical Grade
Trioctylphosphine	Sigma Aldrich	117854	Technical Grade
Trioctylphosphine oxide	Sigma Aldrich	346187	Technical Grade
1-Octadecene	Sigma Aldrich	O806	Technical Grade
Zinc diethyldithiocarbamate	Sigma Aldrich	329703	-
Oleic acid	Sigma Aldrich	364525	Technical Grade
Triethylamine	Sigma Aldrich	471283	-
Cadmium oxide	Alfa Aesar	33235	-
Hexadecylamine	Alfa Aesar	B22459	Technical Grade
1-Dodecylphosphonic acid	Alfa Aesar	H26259	-
Selenium powder	Acros	19807	-
Chloroform	Sigma Aldrich	366919	-
n-Hexane	Sigma Aldrich	208752	-
Microscope slides	VWR	631-0137	Thickness No. 1