Methods for immobilization of DNA on gold surfaces?

03 April 2023 5 2K Report

I've been looking for methods for immobilizing 5'-Thiol modified dsDNA on gold slides. Most of what I've come across suggests using DTT followed by a desalting step or using TCEP. However, I haven't had any luck reducing and then conjugating the DNA onto the gold slides. I've seen some methods use DTT1,2, and some use TCEP3, and some include an incubation step with MCH2,3 after the thiol reducing step. It's been difficult finding a complete and satisfactorily detailed method online.

The DNA I am using is a 1 kb long double-stranded molecule with a 5'-Thio modification at one end and a 5'6-FAM modification at the other end. I need to synthesize the strands myself via PCR using two modified primers and a template strand rather than ordering the fully modified 1 kb strand, so my working concentrations are usually fairly low (~70-100 ng/uL after PCR and spin-column purification).

Can anyone provide a detailed method that has worked for them for reducing thiol modified DNA and immobilizing it onto gold surfaces or gold monolayers?

Hegner, M., Wagner, P. & Semenza, G. Immobilizing DNA on gold via thiol modification for atomic force microscopy imaging in buffer solutions. FEBS Letters 336, 452–456 (1993).

Ladik, A. V., Geiger, F. M. & Walter, S. R. Immobilization of DNA onto Gold and Dehybridization of Surface-Bound DNA on Glass. 5.

Das, J. et al. Reagentless biomolecular analysis using a molecular pendulum. Nat. Chem. 13, 428–434 (2021).

Joong Hyun Kim

TCEP works very well as much as DTT without desalting. For details, please refer to this Article Improving Pb(2+) detection using DNAzyme-based fluorescence ...Article Improving Pb(2+) detection using DNAzyme-based fluorescence ...

for details. However, I have not tried 1kb long DNA with TCEP. In the case of a planar gold surface, you might not need desalting or salt aging. Why don't you provide us with the detailed protocol you made? Then we might find a better solution

Rory Majule

Joong Hyun Kim thank you for the direction. At the current point in our research, we're just trying to confirm that we can bond our DNA to a gold surface. We are using a strand ~1kb long amplified using a modified forward primer with 5' FAM fluorescein (https://www.idtdna.com/site/catalog/modifications/product/1108) and a modified reverse primer with a 5' Thio C6 S-S group (https://www.idtdna.com/site/catalog/modifications/product/1681). After PCR amplification, I use a Monarch PCR & DNA Cleanup Kit (https://www.neb.com/products/t1030-monarch-pcr-dna-cleanup-kit-5-ug#Product%20Information) to purify the samples. To make sure it's working correctly, we're trying to bind the dsDNA to a gold plated microscope slide (https://substratasolutions.com/product/gold-coated-microscope-slides-au-1000-a-10-pkg/) and then visualize the fluorescence under the microscope. Here are the four experiments I've tried so far, plus a couple of additional questions I've included at the bottom:

Reduction using DTT:

Prepare a 20 mM solution of DTT in 1X TE, pH 8. For 5 mL, dissolve 15.4 mg of DTT in buffer.

Mix 80 uL 20 mM DTT with 20 uL purified DNA sample.

Incubate at room temp for 1 hr.

Re-suspend MicroSpin G-25 column resin (https://www.sigmaaldrich.com/US/en/product/sigma/ge27532501) in the columns by vortexing.

Loosen the cap one-quarter turn and twist off the bottom closure.

Place the column in the supplied Collection tube for support.

Spin for 1 min at 3000 rpm (~735 x g).

Place the column into a fresh 1.5 mL microcentrifuge tube.

Slowly apply 50 uL of sample to the top-center of the resin, being careful not to disturb the resin bed.

Spin for 2 min at 3000 rpm (~735 x g). The purified sample is collected in the bottom of the 1.5 mL microcentrifuge tube.

Pipette ~10 µL of sample onto a clean gold slide.

Incubate samples on slide for 24 hours at room temperature.

Wash slide with DI water and dry with compressed air. View under microscope with blue light setting.

Results: In open air, droplets dried completely within a few hours after beginning incubation. After washing and drying the next day, fluorescent specks (likely dust) were observed, but no clear patches where DNA had bound. After more vigorous washing, these specks disappeared as well.

Reduction using TCEP (attempt 1):

Add 5 uL 0.1 mMTCEP, pH 7, to 5 uL purified DNA samples.

Incubate at room temperature for 2 hr.

Pipette ~10 µL of sample onto a clean gold slide.

Incubate samples on slide for 24 hours at room temperature.

Wash slide with DI water and dry with compressed air. View under microscope with blue light setting.

Results: Same as above. In open air, droplets dried completely within a few hours after beginning incubation. After washing and drying the next day, fluorescent specks (likely dust) were observed, but no clear patches where DNA had bound. After more vigorous washing, these specks disappeared as well.

Reduction using TCEP (attempt 2):

7.5 µL 0.1 mM TCEP into 7.5 µL purified DNA samples

Incubate at room temp in the dark for 2 hours

Place silicone sheet with small hole punched into center onto clean gold slide. This will be used to create a gasket seal to prevent evaporation of the droplet.

Pipette DNA+TCEP solutions the gasket hole onto the gold surface.

Place a plastic slide over the hole, sealing the droplet off from air, and secure in place with clips.

Incubate for 42 hrs in the dark at room temp.

Wash slide with DI water and dry with compressed air. View under microscope with blue light setting.

Results: Droplets remained in liquid phase for the duration of incubation. After vigorous washing and drying, no fluorescence was observed.

Reduction using TCEP (maleimide-FAM control experiment):

As an additional control experiment, I used maleimide-FAM (https://www.lumiprobe.com/p/fam-maleimide-6?gclid=Cj0KCQjwla-hBhD7ARIsAM9tQKu1moDNhQRA1b0KiusT3WRa1oys29W227RuZJTwaxY4RpQO3TnE8OoaAjsNEALw_wcB) to check whether my thiol modified samples were actually being reduced. I used a dsDNA sample identical to the one described above, only it lacked the 5' FAM modification. After TCEP reduction and FAM-Maleimide conjugation, this normally non-fluorescent 5' Thio modified dsDNA should show up as a fluorescent band if I run it in a gel, even if I don't include any dyes or stains like SYBRsafe.

Disolved DNA samples in degassed 1x PBS, ~pH 6.5

Add 1.6 µL 40 µM TCEP to 18.4 µL purified DNA sample (~20 ng/µL), and vortex. This was calculated out to be roughly 100x molar excess of TCEP.

Incubate in the dark at room temperature for 2 hours.

Add together 8.4 µL 5000 nM FAM-Maleimide (in DMSO), 1.6 µL DMSO, and 20 µL reduced DNA sample. Gently vortex. This was calculated out to be roughly 20x molar excess of FAM-Maleimide.

Flush with argon gas and close caps.

Protect from light and incubate at 4˚C overnight

Run in 1.5% agarose gel using TBE buffer.

Results: Inconclusive. Positive control bands using strands that already had a FAM modification were clearly visible, regardless of whether they had undergone reduction and conjugation. Due to fluorescent blowout from the FAM-Maleimide, it was difficult to determine whether a faint band was present in the lane with the 5' Thio modified strand that had undergone reduction and conjugation.

Additional Questions:

Is it best to let reduced samples in solution dully dry out on the gold surface during incubation? Or should they be kept from evaporating? Or should they be desiccated and then rehydrated as described in the 1993 Hegner paper cited in the original post?
Is the addition of an MCH incubation step critical for achieving robust attachment? Or is it just included to remove non-specifically bound DNA prior to analysis?

Joong Hyun Kim

Is it usual to amplify DNA with thiolated Primer? Otherwise, you may need to check how thiolated primer affects PCR. I am not sure but thiolated primers might form di-thiol between themselves.

The immobilizing DNA amount might not be enough ( I did not check it).

During incubation of your DNA on the gold surface, it should not be dried. TCEP makes the solution acidic. I recommend you need to mix TCEP and DNA in a proportionate buffer. Usually, I used PB as the article. I am not sure that such long incubation. Anyhow, if you add salt such as NaCl to the incubating solution. It helps to increase the immobilized density of DNA because It reduces electrostatic repulsion between DNA. You might refer to these two articles Article Aptamer duo-based portable electrochemical biosensors for ea...

and 10.3390/bios10050045.

Good luck

Fang Xie

Hi, Rory! I am not sure the process of your DNA synthesis, but I did the modification of DNA on gold electrode. DNA I used is purchased from technology company. Maybe the following details can help you. 10 uL DNA(40-base, 10 uM, pH 7.33, 10 mM PBS+15 mM NaCl solvent) + 10 uL TCEP(dissolved immediately, 2 mM, pH 7.33, 10 mM PBS+15 mM NaCl solvent) mixed then incubated for 2h at 27℃ in centrifugetube (in dark). Then 80 uL 10 mM PBS+15 mM NaCl was added to dilute. The polishied gold electrode was moved into the tube and sealed the tube by parafilm. Then I inverted the electrode so that the DNA solution dipped on gold surface. Usually I incubate the electrode for 1 night at room temp (in dark).

Mridu Sharma

1. TCEP is much easier to work with.

2. I think the amount of DNA you are using must be at least in micromolar concentration.

3.TCEP:DNA ratio is 1:10

4. You should include a salt aging step to increase the density of DNA strands on the gold film. Salt aging should take 12-24 hours.

How these DNA strands and hence the dye molecules spread along the gold film is an important point.

We have an optimized protocol for conjugation of DNA strands with gold nanostructures. It might help you.

All the best.

Kaur, Vishaldeep, Mridu Sharma, and Tapasi Sen. "DNA Origami-Templated Bimetallic Nanostar Assemblies for Ultra-Sensitive Detection of Dopamine." Frontiers in Chemistry 9 (2021): 772267.

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