Colorimetric Detection of Aliphatic Alcohols in β-Cyclodextrin Solutions

The sensitive, selective, and practical detection of aliphatic alcohols is a continuing technical challenge with significant impact in public health research and environmental remediation efforts. Reported herein is the use of a β-cyclodextrin derivative to promote proximity-induced interactions between aliphatic alcohol analytes and a brightly colored organic dye, which resulted in highly analyte-specific color changes that enabled accurate alcohol identification. Linear discriminant analysis of the color changes enabled 100% differentiation of the colorimetric signals obtained from methanol, ethanol, and isopropanol in combination with BODIPY and Rhodamine dyes. The resulting solution-state detection system has significant broad-based applicability because it uses only easily available materials to achieve such detection with moderate limits of detection obtained. Future research with this sensor system will focus on decreasing limits of detection as well as on optimizing the system for quantitative detection applications.


MATERIALS AND METHODS
The analytes and dyes were obtained from Millipore Sigma chemical company and the cyclodextrins were obtained from Tokyo Chemical Industry chemical company, and all chemicals were used as received. Samples were illuminated using the homemade lightbox detailed on the following page. All photos were taken with a Samsung Galaxy S8+ (model number: G950U) on manual mode with the following settings: ISO set to 100, aperture set to 1/350, macro focused (close-up focus), and the white balance set at 5500K. Images were cropped using software from https://www.birme.net. Fluorescence measurements for the binding experiments were performed using a Shimadzu RF 6000 spectrophotometer. Both the excitation and emission slit widths were set to 3.0 nm. All fluorescence spectra were integrated vs. wavenumber on the X-axis using OriginPro 2019 Version 9.60. All arrays were generated using SYSTAT Version 13.1. Figure S1: Structure of alcohol analytes and highly colored dyes All analytes were used as received. The dyes were prepared at a concentration of 1.0 mg/mL in isopropanol. Diluted dye solutions were prepared by diluting 5.0 mL of the concentrated stock solutions with 150 mL of DI H2O. The final concentrations of the dyes are shown in Table S1, below.

EXPERIMENTAL DETAILS FOR CYCLODEXTRIN SOLUTIONS
The method for making the cyclodextrin solutions was taken from the previous work of high school student Priva Halpert working under the supervision of Dr. Levine. As a high school student working remotely, Halpert based her measurements on volume of each component, using teaspoons, cups, etc. In her procedure, 0.5 teaspoon of cyclodextrin was added to 0.25 cups (59.15 mL). We wanted to replicate her procedure and converted her measurement of 0.5 teaspoons to grams for each cyclodextrin and scaled up to make a solution with a final volume of 250 mL. These conversions are summarized in the table below, together with the final concentrations of the cyclodextrins.

EXPERIMENTAL DETAILS FOR CROPPING PHOTOGRAPHS AND RGB MEASUREMENTS
All photographs were cropped using the online tool found at https://www.birme.net. Photos from the same trial were uploaded to the site then cropped to a 500x500 pixel ratio ( Figure S6). The cropped photos were then opened into the ImageJ software. These measurements were recorded using the RGB measurement plug-in provided in the software ( Figure S7). Figure S6. Cropping sample photos to 500x500 pixel ratio using software on https://www.birme.net Figure S7. Processing of cropped sample photo using the ImageJ software RGB Measurement plug-in.

EXPERIMENTAL DETAILS FOR THE OPTIMIZATION OF THE SUPRAMOLECULAR CYCLODEXTRIN HOST
In a glass sample jar, 10.0 mL of β-cyclodextrin stock solution was combined with 10.0 mL of one of the diluted dye solutions. This mixture was manually shaken for 1 minute to ensure a homogeneous mixture. After mixing, 5.0 mL of alcohol was added. This mixture was transferred to the sample cup and placed in the lightbox. The cover was placed on and a photo was taken using the smartphone with the settings detailed above. This procedure was repeated for methyl-β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin with both dyes and each of the three alcohols (18 total samples). These samples were replicated 4 times in total with an average standard deviation in RGB values of 0.70%. Full summary tables of standard deviation and standard deviation percentages can be seen on page S15. As a control, experiments were also conducted in the absence of cyclodextrin but under otherwise identical conditions. Summary tables and figures from these experiments are included herein.

EXPERIMENTAL DETAILS FOR THE OPTIMIZATION OF ANALYTE CONCENTRATION
In a glass sample jar, 10.0 mL of 2-hydroxypropyl-β-cyclodextrin stock solution was combined with 10.0 mL of one of the diluted dye solutions. This mixture was manually shaken for 1 minute to ensure a homogeneous mixture. A 0.5 M solution of the alcohol was made by adding the corresponding amount of alcohol to the cyclodextrin-dye solution in the glass jar, with additional samples tested for each alcohol at a variety of concentrations (0.5 M, 1.0 M, 2.0 M, and 3.0 M) using both BODIPY and Rhodamine dyes (8 samples replicated 3 times each). The volume of alcohol necessary to obtain an 0.5 M solution is seen in Table S3, below. These solutions were transferred to a sample cup, placed in the lightbox, and a photo was taken of each.

EXPERIMENTAL DETAILS FOR LIMIT OF DETECTION EXPERIMENTS
The limit of detection (LOD), defined as the lowest concentration of the analyte that can be detected, was obtained using the calibration curve method, following procedures reported by Loock et. al. The limit of quantification (LOQ) is the lowest concentration of analyte that can be quantified. The limit of detection 1 and quantification 2 experiments were conducted following literature-reported procedures.
To determine the LOD and LOQ, each dye-analyte combination in 2-hydroxypropyl-β-cyclodextrin solution was examined in the following manner: 1. In a glass sample jar, 10.0 mL of 2-hydroxypropyl-β-cyclodextrin stock solution was combined with 10.0 mL of one of the diluted dye solutions. This mixture was manually shaken for 1 minute to ensure a homogeneous mixture.
2. The solution was transferred to a sample cup and subsequently placed in the light box. A photo was taken in order to obtain the blank measurement of the solution (i.e. in the absence of any alcohol, before any analyte had been added).
3. Using a 20-200 μL Fisherbrand Elite micropipette, 100 μL of an alcohol was added and a picture was taken. These 100 μL additions continued until 6.0 mL of alcohol was in solution.
5. Photos were cropped to a 500x500 pixel ratio centered on the center of the sample cup using software from https://www.birme.net and the RGB values of the photos were measured using the RGB measurement plug-in tool in the ImageJ software.
6. The Green values (Y-axis) were chosen to be plotted verses the molarity (X-axis), because they exhibited the most consistent trends. Calibration curves were generated, fitted with an exponential function and an equation was determined.
7. The limit of the blank is defined according to the following equation: where m is the average of the values obtained from the blank sample and SD is the standard deviation of those measurements.
8. The limit of the blank was entered as the y-value in the equation from step 6, and the corresponding xvalue was calculated. This value was the LOD of the system in M.
9. The LOQ was determined in a similar procedure to the LOD. The limit of quantification blank is defined according to the following equation: This value is then inputted as the y-value in the equation from step 6, and the corresponding x-value was calculated. This value is the LOQ for the system in M.

EXPERIMENTAL DETAILS FOR BINDING CONSTANT EXPERIMENTS
Fluorescence measurements were performed on a Shimadzu RF 6000 spectrophotometer. Both the excitation and emission slit widths were set to 3.0 nm. A 1.45 mM solution of 2-HP-β-CD was prepared in DI H2O. A solution of 2.09 mM dye 5 was prepared in DI H2O and a 2.54 mM solution of dye 4 was prepared in tetrahydrofuran, with the solvent selection and concentration optimized based on concentration. In both binding experiments, 2.50 mL of water was added to quartz cuvette. In the dye 5 trial, 8 μL of the dye was added. In the dye 4 series, 10.5 μL was added. The tip of the micropipette was used to stir the solution in the cuvette to ensure a homogeneous mixture. Fluorescence measurements of these were taken 4 times. In each trial, 1 μL of the 2-HP-β-CD solution was added to the cuvette and the same scanning process was completed. This was repeated for the total addition amounts seen on page S19 in Tables S10 and S11, the total addition amount representing the concentration at which the observed signal plateaued. All fluorescence spectra were integrated vs. wavenumber on the Xaxis using OriginPro 2019 Version 9.60. Binding constants (Ka) were determined using the equation shown below: where F is the fluorescence of the sample, F0 is the fluorescence of the blank, [G] is the concentration of the guest which in this case is 2-HP-β-CD, and Ka is the binding constant. The average Ka was determined to be 3.32 x 10 5 M -1 and 1.59 x 10 5 M -1 for dyes 4 and 5 respectively.

EXPERIMENTAL DETAILS FOR ARRAY GENERATION EXPERIMENTS
Array analysis was performed using SYSTAT 13 statistical computing software with the following settings: The results are summarized in the tables, starting on the page S20, and in the figures, beginning on page S22.

EXPERIMENTAL DETAILS FOR COMPUTATIONAL MODELING
Spartan '18 was used to calculate the equilibrium at ground state in the gas phase, using a semi-empirical PM3 model for each analyte ( Figure S8). This allowed an electrostatic potential map surface to be overlaid on the molecules. Molecular Operating Environment 2018 (MOE) was used to do the docking studies for each dye and 2-HP-β-CD. A general energy minimization was performed using the "quick prep" function and the default settings. For the docking studies, the set of atoms defined as the receptor was both 2-HP-β-CD and the solvent so that the dye could move freely in the system. Placement was done using the Triangle Matches method with London Dispersion dG score in 30 poses. Refinement was done using the Rigid Receptor method with GBVI/WSA dG score in 5 poses ( Figure S9). This generated the docking of the dyecyclodextrin complex with the lowest energy confirmation.

SUMMARY TABLES FOR BINDING EXPERIMENTS
2-HP-β-CD was added into the cuvette with the dye solution. Sequential additions were conducted, and were stopped when the fluorescence measurements begin to plateau. Full experimental details can be seen on page S11, above.  Tables of Cyclodextrin-Analyte Combinations with Dyes 4 and 5 Arrays   Table S13. Analytes with β-CD and Dye 4 Table S14. Analytes with Me-β-CD and Dye 4 Table S15. Analytes with 2-HP-β-CD and Dye 4 Table S16. Analytes with β-CD and Dye 5 Table S17. Analytes with Me-β-CD and Dye 5

SUMMARY FIGURES FOR ALL LOD EXPERIMENTS
The red lines shown on each graph are representative of the lines of best fit of the equations given on page S18 in Table S9.