Visual Readout – Gold Nanoparticles

Results

To achieve the desired in-field usability of our test system, we looked into a variety of visual readout systems, which utilize the unspecific, single-strand trans cleavage activity of our activated Cas12a-ribonucleoprotein complex, upon activation through recognition of the target pathogen sequence. In the end we mainly focused on the usage of gold nanoparticles (AuNPs), because of their variable optical properties and their stable functionalization potential, especially via sulfur-gold-bonds [1]. Therefore, we introduced reduced thiolated ssDNA (SH-DNA) to covalently attach to the AuNPs and achieve a controlled aggregation of AuNPs through the addition of a ssDNA-linker molecule, as the substrate for the Cas12a-cleavage induced color change and visual readout capabilities. A schematic overview of the functionalization process is shown in Figure 1

A beautiful sunset over the mountains
Figure 1: Schematic of experimental procedure of gold nanoparticle (AuNP) functionalization with single stranded thiolated-DNA-oligonucleotides, the linker-based aggregation and the spectrophotometric assay for qualitative color differentiation.

Variations of functionalization protocol

The basis of our functionalization procedure was adapted from Guo et al. (2023) [2]. A critical point during the process is the successful attachment of thiolated ssDNA-oligos to the gold nanoparticle (AuNP) surface, since incomplete or insufficient functionalization prevents the controlled aggregation of particles upon linker addition. To improve the robustness of oligo attachment, and utilizing the engineering design cycle, several variations were introduced at different stages of the protocol. 1) We increased the duration of the TCEP reduction step to 2 h, aiming to ensure complete cleavage of disulfide bonds and maximize availability of reactive thiol groups. The attachment process was monitored by measuring the ssDNA concentration that was still present in the supernatant, after each step of the functionalization process. 2) We tested an exchange of the original citrate buffer of the AuNPs for a weak phosphate buffer. This was intended to provide a more stable ionic environment during conjugation. While we did not observe any visual changes regarding the colloidal stability, the buffer exchange process has led to loss of AuNPs and by this visual intensity following the regular protocol. 3) We added an ethanol washing step after the reduction process to remove residual TCEP, which can interfere with Au–S bond formation. 4) We elongated the salt-aging steps, since multiple studies have highlighted this as the most critical phase for achieving high-density DNA loading on AuNPs [3]. Extended salt-aging was expected to promote gradual adaptation of the particles to high ionic strength conditions, thereby supporting denser and more stable ssDNA attachment. 5) We varied the combination of SH-DNA pairs, with slight differences within the hybridization of the AuNP-SH-DNA to the linker-DNA. Furthermore, we tested 3 different linker-DNA lengths, to determine at which maximal length, to prevent steric hinderance of the Cas12a-protein, the controlled aggregation still leads to an observable color change. 6) We tested AuNPs with a diameter of 60 nm to potentially achieve even more differentiable optical properties, for that we used a higher initial SH-DNA concentration to reach a similarly dense attachment in comparison to the 10 nm AuNPs. This did not lead to any success, because the AuNPs already destabilized within regular buffer conditions and not controlled after the addition of the linker molecules. These variations have led us to the most robust procedure and can be found in the Materials and Methods section.

Linker-based aggregation of AuNPs

The results of Figures 2-5 were not produced with AuNPs, from the fully optimized functionalization procedure, while Figure 6 shows the spectra from the optimized protocol and thus a robust and clear differentiation of control and linker-aggregated AuNPs. The data behind the figures 2, 3 and 5 (A) is the same and is repeated for the purpose of highlighting different aspects of the sub-experiments.

DNA1/2 vs DNA 1R/2R

In Figure 2, the difference of absorbance after the addition of the linker molecules is shown for the two types of SH-Oligo pairs (DNA 1/2; DNA 1R/2R) that were attached to the AuNPs is shown. For every linker-combination the Ratio of Absorbance (A520/A560) is calculated, to have a qualitative indicator of the appearing change in color. The control samples are always the mixed DNA-functionalized AuNPs, NaCl, but no linker molecules.

A beautiful sunset over the mountains
Figure 2: (A) Spectrophotometric absorbance assay (520, 540, 560 nm) of AuNPs attached with SH-DNA 1 and SH-DNA 2, mixed with linker-ssDNA molecules of three different lengths at 750 mM NaCl concentration. (B) Mixture of SH-DNA 1R and SH-DNA 2R

The control for both SH-DNA attachments shows its absorbance peak at 520 nm and an A520/560-ratio of 1.4, which is associated with a reddish-pink color, while the lower ratios of ~1.1 and a shifted peak to 540 nm, are associated with a more transparent and violet appearance, which speaks for successfully aggregated AuNPs. Even though the linker-based aggregation seems to be similarly successful for both SH-DNA pairs, we continue the following tests with the DNA 1/2 pair, because even linker 3, which theoretically would offer the most space for the Cas12a, shows a observable shift in absorbance.

No wash vs. Ethanol wash

As previously mentioned, to prevent any unintended effect of remaining reducing agents, we tried to implement an ethanol washing step, after the initial reduction. The results of the spectrophotometric analysis of the AuNPs which were functionalized after the washing step, can be observed in Figure 3B.

A beautiful sunset over the mountains
Figure 3: Comparison of spectrophotometric assay of AuNPs prepared without (A) and with (B) an Ethanol washing procedure after the reduction of the SH-DNA. Both

In comparison to the spectrophotometrically determinable color shift of the non-washed AuNPs, the A520/560-ratio of the washed particles, even the control, has dropped uniformly (Figure 3). Furthermore, a general drop in absorbance for the ethanol washed AuNPs was observed, due to a certain loss of SH-DNA during the washing steps, resulting in a less dense attachment to the AuNPs, which leads to a worse colloidal stability of the individual AuNPs within the buffer. This premature aggregation, because of the insufficient stabilization of the SH-DNA also seems to be the reason for the color shift, even for the control sample. The optical properties of the linker-aggregated and the control, between the ethanol washed and unwashed AuNPs are shown in Figure 4.

A beautiful sunset over the mountains
Figure 4: Visible color change after linker addition to AuNPs from no wash and ethanol wash batches.

Comparison of Linkers

In Figure 5 the difference in absorbance regarding the linker length was assessed, with 1 being the shortest and 3 being the longest.

A beautiful sunset over the mountains
Figure 5: Comparison of spectrophotometric absorbance of AuNP-DNA 1/2 mixtures in combination with three linkers of different lengths.

While the absorbance ratio indicates that a color change for all linkers has appeared, the visual observation was not able to confirm this. Potentially because of the observed, spectral-wide drop in absorbance for the linkers 2 and 3 (Figure 5). This result potentially carries the risk of steric hindrance for the Cas12a cleavage activity and thus an insufficient color change.

Optimized protocol triplicates of linker 1 10 min and 4 h after linker addition

To confirm the protocol that our optimization cycle has led us to, we performed one complete fresh batch of functionalized AuNPs, that were tested as triplicates in presence of linker 1, as the most successful candidate.

A beautiful sunset over the mountains
Figure 6: Spectrophotometric (4480-580 nm) triplicate measurements of AuNP mixtures (SH-DNA 1/2) functionalized with the optimized protocol and combined with the linker 1. (A) 10 min after linker addition. (B) 4 h after linker addition.

After 10 min, the linker-based aggregation of the AuNPs is very uniform for the triplicates and clearly differentiable by the absorbance ratio and the whole spectrum to the control (Figure 6A). But after 4 h this difference shrinks, which might indicate the general destabilization of the functionalized AuNPs, even without the linker, which might carry on the problem of unsatisfying color change, after the cleavage of the linker molecules by Cas12a, after longer storage durations. The visual color change after 4 h is also not as satisfactory, as right after linker addition. On the other side would it be possible to exchange the buffer of the linker-aggregated AuNPs again to a more physiological, that the dispersing after addition of the Cas12a and upon longer storage can be achieved more easily.

Outlook and further optimizations

The goal of upcoming experiments regarding the implementation of the AuNP-based visual readout system will address the achievement of a more intense and easier to differentiate color change, by changing the AuNP concentration, their diameter and the general buffer conditions. Furthermore, our clear goal is to reach the point of a working proof-of-concept, where our presented, separate systems will be combined and after the recognition of the pathogen sequence by Cas12a, the trans cleavage activity will induce the dispersion of the aggregated AuNPs and by that an in-field ready visual readout.

Material and Methods

Functionalization of gold nanoparticles with ssDNA

Gold nanoparticles (AuNPs; 10 or 60 nm, citrate-stabilized; Sigma-Aldrich, Germany) were functionalized with thiolated single-stranded DNA (SH-oligos; IDT) following a salt-aging and washing protocol. SH-oligos (3 nmol, 30 µL of 100 µM) were reduced with tris(2-carboxyethyl)-phosphine (TCEP, 30 mM; Sigma-Aldrich, Germany) at a 100:1 molar ratio in nuclease-free water for 2 h at room temperature. Reduced SH-oligos were then incubated with AuNPs (0.01 nmol in 1 mL) overnight at room temperature under gentle shaking. To stabilize the DNA–AuNP conjugates, sodium phosphate buffer (200 mM, 2 M NaCl, pH 7.4; Carl Roth, Germany) was added in four aliquots of 40 µL at 1 h intervals, reaching a final NaCl concentration of 0.2 M. The mixture was further incubated overnight at room temperature. Excess unbound DNA was removed by centrifugation (12,000 g, 20 min; Eppendorf Centrifuge 5420, Eppendorf, Germany) and resuspension in sodium phosphate buffer (10 mM, pH 7.4). The washing step was repeated three times. Functionalized AuNPs were finally resuspended in 500 µL sodium phosphate buffer (10 mM, pH 7.4) and stored at 4 °C until use.

Visual assay for AuNP aggregation

DNA-functionalized AuNPs (with complementary SH-oligos in both orientations, 50 uL each/well) were mixed with 10 uL linker-ssDNA (molar ratio of 50:1) in 96-well microplates (Greiner Bio-One, UK). 20 uL NaCl (5 M; Carl Roth, Germany) was added to reach a final concentration of 750 mM to reduce the electrostatic repulsion of the negatively charged DNA molecules and thus drives aggregation. The mixtures were incubated for 1 h at room temperature under shaking conditions. Spectrophotometric analysis was performed on a microplate reader (Tecan Life Sciences, Switzerland) by recording absorbance spectra between 480–580 nm. The absorbance ratio at 520/560 nm was calculated to quantitatively assess nanoparticle aggregation and the resulting color change.

Sequences

                                                                                                                                                                       
NameSequence (5‘ – 3‘)
SH-DNA 1SH C6-TTTTTCGCTCCGATCGCTC
SH-DNA 2GGGATCGCTCACCGTTTTT-C3 SH
SH-DNA 1RTTTTTCGCTCCGATCGCTC-C3 SH
SH-DNA 2RSH C6-GGGAGAATTCACCGTTTTT
linker 1GAGCAAGCTTAGCGAAAAATCAAGATACATGAAAAAACGGTGAATTCTCCC
linker 2GAGCAAGCTTAGCGAAAAATCAAGATACATCAAGATACATGAAAAAACGGTGAATTCTCCC
linker 3GAGCAAGCTTAGCGAAAAATCAAGATACATCAAGATACATCAAGATACATGAAAAAACGGTGAATTCTCCC

Notebooks

In this section, the Notebooks are uploaded.

References

[1] Zhou, W., Gao, X., Liu, D., & Chen, X. (2015). Gold Nanoparticles for In Vitro Diagnostics. Chemical Reviews, 115(19), 10575–10636. https://doi.org/10.1021/acs.chemrev.5b00100 [2] Guo, H., Zhang, Y., Kong, F., Wang, C., Chen, S., Wang, J., & Wang, D. (2023). A Cas12a-based platform combined with gold nanoparticles for sensitive and visual detection of Alternaria solani. Ecotoxicology and Environmental Safety, 263, 115220–115220. https://doi.org/10.1016/j.ecoenv.2023.115220 [3] Hurst, S. J., Lytton-Jean, A. K. R., & Mirkin, C. A. (2006). Maximizing DNA Loading on a Range of Gold Nanoparticle Sizes. Analytical Chemistry (Washington), 78(24), 8313–8318. https://doi.org/10.1021/ac0613582