Engineering Moleculaire herkenning met Bio-mimetische Polymers op enkelwandige koolstof nanobuisjes

The overall goal of this procedure is to prepare near-infrared fluorescent single walled carbon nanotubes for molecular sensing that have been functionalized using biomimetic polymers. These nanosensors can enable time resolved imaging in living tissue. For example, they can be used to detect dopamine in the brain.

The main advantage of these sensors is their inherent near infrared fluorescence, which enables imaging deeper into tissue. Synthetic sensors enable the detection of molecules without natural molecular recognition elements making this generic method of sensors development especially useful for small molecule detection. To prepare a nucleic acid suspension of SWNTs, first dissolve nucleic acids in 0.1 molar sodium chloride to a concentration of 100 milligrams per milliliter.

In a fume hood, remove static electricity from the disposable spatula, microcentrifuge tubes, and the SWMT stock, using an antistatic gun. Next, add 20 microliters of the nucleic acid solution to 980 microliters of 0.1 molar sodium chloride. Then add 1 milligram of washed SWNTs to the resulting nucleic acid solution before sonicating the mixture.

Positioning the probe tip during sonication is critical. If foaming occurs, reposition the probe tip, and try lowering the amplitude. Alternatively, you can try pulses of sonication or bath sonication if the suspension remains poor.

Using an ultrasonicator with a 3 millimeter diameter tip, sonicate the solution for 10 minutes at 40%amplitude in an ice bath. Centrifuge the DNA SWNT solution twice for 90 minutes at 16, 100 xg. Collect and keep the supernatant, being careful not to disturb the pellet containing carbon nanotube bundles and aggregates.

Discard the pellet in accordance with institutional hazardous waste procedures appropriate for nanomaterials. Measure the solution absorbance at 632 nanometers using a UV-vis spectrophotometer to determine the approximate concentration of suspended SWNTs, in accordance to Lambert-Beer's law, and the appropriate dilution factor. For amphiphilic polymer suspensions, first remove the static electricity from the disposable spatula, microcentrifuge tubes, and SWNT stock.

In a fume hood, add five milligrams of SWNTs to five milliliters of 2%sodium cholate solution. Using an ultrasonicator with a six millimeter diameter tip, sonicate the solution for one hour at 40%amplitude in an ice bath. Centrifuge the sample at 150, 000 xg for four hours using an ultracentrifuge, before carefully collecting the supernatant.

Next, dissolve 1%weight of amphiphilic polymer in the scSWNT solution. Then, dialyze the polymer scSWNT solution using a 3.5 kilodalton dialysis membrane against one liter of deionized water or buffer for five days. Change out the water or buffer at hour two, and at hour four.

Measure the solution absorbance at 632 nanometers using a UV-Vis spectrophotometer to determine the approximate concentration of suspended SWNTs. To prepare the BSA-biotin coated slides, clean a microscope slide and 0.17 millimeter cover glass with deionized water, followed by methanol, acetone, and a final rinse of deionized water. Create channels by placing several pieces of double sided tape approximately five millimeters apart on the clean microscope slide.

Seal the channels by taking a long glass cover slip and pressing it onto the top of the double sided tape. Be sure to center it on the microscope slide so that the edges of the cover slip and slide are not flush. Next, add 100 microliters of BSA-biotin stock solution to 900 microliters of 1x Tris buffer to a final concentration of one milligram per milliliter.

Flow 50 microliters of the BSA-biotin solution into the channel by pipetting the solution into one end and wicking away the solution at the other end using a tissue. After incubating the solution for five minutes, perform three to five flushes with 50 microliters of 0.1 molar sodium chloride. Next, dilute 40 microliters of five milligrams per milliliters NAV stock solution into 960 microliters of 1x Tris buffer, to a final concentration of 0.2 milligrams per milliliter.

Flow 50 microliters of the NAV solution into the channel by pipetting the solution into one end and wicking away the solution at the other end using a tissue. After incubating the slide for two to five minutes, perform three to five flushes with 50 microliters of 0.1 molar sodium chloride. Then, dilute the stock solutions of suspended SWNTs and imaging buffer to a concentration of 1 to 10 milligrams per liter.

Flow 50 microliters of this solution into the channel and incubate for five minutes. Gently rinse away excess SWNTs using 50 microliters of imaging buffer. To measure the reversible response of GT15 DNA-SWNTs to dopamine, first mount the sensor coated channels onto the fluorescence microscope stage.

Bring the channels into focus using a 100X Oil Objective and indium gallium arsenide camera. Then, add 50 microliters of 100 micromolar dopamine solution in phosphate buffered saline to the flow channel, and record the change in fluorescence intensity. Wash out the dopamine solution using phosphate buffered saline, and observe the change in fluorescence of the individual SWNT sensors.

To perform the well plate screening for analyte response of biomimetic polymer SWNTs, pipette equal volumes of the suspended SWNT sensor into each well. Use enough volume to cover the bottom of the well uniformly. Then, pipette analytes from the screening library into the well plates.

Prepare each analyte in triplicate to account for potential well to well variation and fluctuations of excitation intensity, or temperature. Raise the objective while monitoring the emissions spectra using the spectrometer and indium gallium arsenide array. Optimal position of the objective will put the focal plane approximately in the middle of the sample volume in the well, which should correspond to a maximum in measured intensity.

For each sample well, record a one to 10 second exposure to collect a spectrum. Compare the emissions spectra for each well to a control containing just SWNTs without additional analyte, to quantify the fluorescence response. Absorbance spectrum of GT15 DNA nanotube sensors shows multiple peaks corresponding to well dispersed carbon nanotubes of different chirality.

The near infrared fluorescence of GT15 DNA nanotube sensors is shown. Interestingly, fluorescence intensity approximately doubles in response to dopamine. In this figure, near infrared fluorescence microscopy reveals single GT15 DNA nanotube sensors immobilized on the surface of a glass cover slip.

Moving forward it is important to test the sensor for in vivo use, for example expanding the analyte library to include molecular competitors, testing for linearity of response in the biologically relevant concentration regime, and also testing for reversibility. Once mastered, this approach can be used to make a library of different synthetic sensors from molecules without known molecular recognition elements.