Hexanols

Aldehydes, acetal, methanol, esters and higher alcohols (also known as fusel alcohols) were quantified by GC-FID. Twenty-one compounds were determined following two different procedures. In both cases, the equipment used was an Agilent 7890B Gas Chromatograph (Agilent Technologies, Santa Clara, CA, USA) coupled with Flame Ionization Detector.
For the analysis of acetaldehyde, acetaldehyde—diethyl acetal, methanol, ethyl acetate, n-propyl alcohol, 2-butyl alcohol, isobutyl alcohol, n-butyl alcohol, 2-methyl-1-butanol and 3-methyl-1-butanol, the samples were injected in a split mode (split 1:46, 250 °C) into a DB-624 (30 m × 250 µm × 1.4 µm, Agilent Technologies, Santa Clara, CA, USA) column. The oven temperature for the analysis was programmed as follows: 30 °C (30 min), then 6 °C/min to 100 °C (0 min). Temperatures of the injector and the detector were 250 °C and 300 °C, respectively. Nitrogen was used as a carrier at flow of 1.0 mL/min. Data acquisition and analyses were performed using OpenLAB CDS Chemstation (Agilent Technologies, Santa Clara, CA, USA) software.
For the analysis of n-hexanol, 2-phenylethyl alcohol, ethyl lactate, ethyl succinate, ethyl caproate, ethyl caprylate, ethyl caprate, ethyl laureate, ethyl myristate and ethyl palmitate, samples were injected in a splitless mode (1 min, 250 °C) onto CP-WAX 57 CB (25 m × 250 µm × 0.2 µm, Agilent Technologies, Santa Clara, CA, USA) column. The oven temperature program during analysis was as follows: 45 °C (20 min), then 3 °C/min to 170 °C (20 min). Temperatures of the injector and the detector were 250 °C and 300 °C respectively. Nitrogen was used as the carrier gas at a flow of 1.3 mL/min. The data acquisition and analyses were performed using OpenLAB CDS Chemstation (Agilent Technologies, Santa Clara, CA, USA) software.
Standards were made in an ethanol/ultrapure water solution at 40%vol. The linear standard curve of 3-methyl-1-butanol ranges from 1 to 250 mg/100 mL of 100% vol. alcohol. The linear standard curve of methanol, ethyl acetate, n-propyl alcohol, isobutyl alcohol and 2-methyl-1-butanol ranges from 1 to 100 mg/100 mL of 100% vol. alcohol. The linear standard curve of acetaldehyde and acetaldehyde—diethyl acetal ranges from 1 to 50 mg/100 mL of 100% vol. alcohol. The linear standard curve of ethyl lactate ranges from 0.5 to 25 mg/100 mL of 100% vol. alcohol. The linear standard curve of 2-butyl alcohol, n-butyl alcohol, n-hexanol, 2-phenylethyl alcohol, ethyl succinate, ethyl caproate, ethyl caprylate, ethyl caprate, ethyl laureate, ethyl myristate, and ethyl palmitate ranges from 0.1 to 5 mg/100 mL of 100% vol. alcohol. The samples were diluted at 40%vol. with ultrapure water and injected in duplicate. The results were expressed in mg of compound per 100 mL of 100% vol. alcohol.

The olfactometer used to deliver odours under the two-photon microscope was the same one used in the EAG experiment. During an imaging session, the odorants of interest (Geosmin 10^–6, Geosmin 10^–3, and 10^–1 IAA) were presented to the bee in a sequence either as a single odour or as mixtures, and the sequence was repeated 10 times. Each stimulus pulse lasted 3 s with a 12 s inter-stimulus interval and an exhaust system quickly removes the odours from the experimental area. For comparison of response strength and width also 3 floral odours were tested with the same sequences (1-nonanol 5·10^–3, acetophenone 5·10^–3, and 3-hexanol 5·10^–3).

Each Au-SPE used was first cleaned with absolute ethanol (99.5%, Panreac) and thoroughly washed with ultrapure water to remove all traces of ethanol and impurities. To assemble the biosensor, first, the Au-SPE was cleaned, followed by immobilization of the thiolated Apt1_RC on its surface. Ultrapure Mili-Q laboratory grade (conductivity < 0.1 µS/cm) was always used, and PBS 1× was prepared freshly from tablets (Amresco, Dallas, USA). The electrode active area was calculated using the Randles-Sevcik equation and found to be 0.202 cm² before cleaning and 0.215 cm² after cleaning.
The aptasensor was assembled in two simple steps, namely (i) immobilization of the probe on the Au-SPE, followed by (ii) blocking of non-specific binding sites. Firstly, a stock solution of thiolated Apt1_RC (1 µM) in phosphate buffer (5 mM MgCl₂ (Riedel-de Haen), in PBS 1×, pH 7.4) was prepared. Before immobilization on the electrode, the stock solution of Apt1_RC was further diluted in phosphate buffer to a working concentration of 0.5 µM and deprotected by incubation with dithiothreitol (DTT, 0.1 M, Sigma Aldrich, Steinheim, Germany) for 30 min at room temperature followed by heating at 95 °C for 5 min. Deprotection disrupts disulphide bonds, and heating makes the aptamer flexible and interactive. The Apt1_RC (5 µL) was then immediately added to the Au working surface and incubated in a glass chamber at room temperature for 2 h. In this step, the aptamer is immobilized via thiol groups at its 5′ end. Subsequently, the Au surface was washed several times to remove all unbound Apt1_RC probes. The Au-SPE was then incubated with 6-mercapto-1-hexanol (MCH, 5 mM, TCI Chemicals, Zwijndrecht, Belgium) for 2 h at room temperature to block unoccupied sites on the Au surface [32 (link)]. This step was crucial to prevent non-specific binding of biomolecules present in a sample to the Au surface. All steps of the aptasensor assembly were monitored by CV and EIS assays to confirm the modifications created to the Au-SPE. For this purpose, 80 µL (5 mM) of the redox probe was added to an Au-SPE that had been modified, followed by recording of CVs and EIS Nyquist plots of the redox probe.