Visual Detection of SNP in circulating tumor DNA using gold nanoparticles -

Visual Detection of SNP in circulating tumor DNA using gold nanoparticles

Detecting single-nucleotide polymorphisms (SNPs) in circulating tumor DNA (ctDNA) is tough due to the small DNA pieces (about 150 nucleotides) and the high levels of normal cell free DNA (cfDNA) in the background. They were created a quick and simple method using centrifugation and colour changes with gold nanoparticles (AuNPs- assisted colorimetric assay using gold nanoparticles). This method, combined with isothermal amplification, identifies a specific SNP (G to C mutation) in the KRAS gene, specifically the p.G13D mutation, within ctDNA. Their approach is different from traditional AuNP aggregation assays and incorporates four unique design concepts.

Firstly, they add a centrifugation step at the end of the process to enhance the visual detection of the SNP ctDNA by causing distinct colour changes through precipitation. Secondly, to meet the high demand for clinical use, they use a concept called “critical linker concentration" to speed up the reaction. Thirdly, to clearly distinguish the SNP ctDNA from normal cfDNA and control samples without DNA, they employ a “colour code conversion" strategy by introducing a complementary sequence in the linker DNA to control AuNP aggregation. Finally, they were using ethylenediaminetetraacetic acid at room temperature to deactivate enzymes and stabilize the AuNP solution, preventing unwanted aggregation.

Materials:

Gold nanoparticles (AuNPs) of approximately 13 nm were made using a standard citrate reduction method in water. Various chemicals and enzymes, including Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), Tris–HCl buffer, ethylenediaminetetraacetic acid (EDTA), CaCl2, MgCl2, HCl, NaOH, NaCl, and Exonuclease III (ExoIII), were obtained from Sigma-Aldrich, Merck, and New England Biolabs. Buffers, such as enzyme reaction buffer, HEPES buffer, PBS buffer, and elution buffer, were prepared or purchased accordingly. DNA oligonucleotides, including “wtKRAS" and “mtKRAS" representing normal and mutated cfDNA, “mLinker" for mutation detection, “OA" and “OB" for AuNP conjugates, and “c-mLinker," “c-OA," and “c-OB" for colour conversion, were obtained from IDT Company.

Preparation of single stranded DNA–nanoparticle (ssDNA–NPS):

To create ssDNA–NPs, disulphide-protected OA or OB ssDNA was activated using TCEP, then purified and quantified. Activated ssDNA was mixed with TCEP, AuNP solution, and HEPES buffer. After incubation, pH was lowered with HCl, and salt concentration increased with NaCl. After further incubation and neutralization with NaOH, ssDNA–NPs were purified by centrifugation, and their DNA surface density was determined. The purified ssDNA–NPs were stored in PBS buffer at 4 °C.

Sandwich ssDNA–NP assembly and determination of “critical linker concentration.

For the sandwich ssDNA–AuNP assembly, equal amounts of ssDNA–NP conjugates and mLinker probe were mixed in PBS buffer and incubated at room temperature for 30 minutes. The “OA" and “OB" are disulphide-protected ssDNA used to form pairs with the mLinker on AuNPs. This assembly was used to determine the critical linker concentration by measuring the ratio of absorbance at 700 and 520 nm (A700/A520) relative to the reference DNA–NPs without mLinker. Visually noticeable precipitation occurred after brief centrifugation at 6000 rpm for around 10 seconds.

Assay procedures:

The assay involves five steps: (1) DNA annealing by mixing mLinker probe with wtKRAS or mtKRAS, heating to 95 °C for 10 minutes, and cooling to room temperature. (2) Enzymatic amplification by adding ExoIII and incubating at 37 °C for 1 hour. (3) Enzyme inactivation with EDTA. (4) Color code conversion by adding c-mLinker and allowing hybridization. (5) AuNPs assembly for ctDNA detection by adding PBS buffer and ssDNA–NPs, incubating for 30 minutes, and characterizing with UV-vis. Colloidal stability of ssDNA–NPs with EDTA was studied by mixing with different buffers and recording UV-vis spectra and camera images after 30 minutes of incubation.

Detection of mtKRAS in wild type cfDNA background:

To assess the assay’s clinical relevance, we created a sample mixture with varying percentages of mutated KRAS (mtKRAS) from 0% to 100%. We compared the signals of these mixtures to a control sample with no KRAS. All circulating tumor DNA (ctDNA) samples were diluted in elution buffer (AVE buffer, Qiagen), the same buffer used for cleaning and purifying DNA from clinical patient samples.

Instrumentations and methods:

UV-vis absorbance measurements were conducted using BioTek platereader (Synergy 2, Multi-Mode Reader), Tecan platereader (M200), or Shimadzu UV 2450 Spectrophotometer. Standard 384-well plates (PS, transparent, Corning®) were used for absorbance measurement on all machines, with background signal subtraction before each sample spectrum. Images of DNA–Au NP solutions were captured using a mobile camera (Apple iPhone) after brief spinning at 6000 rpm (∼10 s) in a portable microcentrifuge (Cole-Parmer Personal Microcentrifuge).

Results and discussion:

The SNP ctDNA assay involves five major steps: (1) annealing of the linker probe and target DNA, (2) enzymatic amplification reaction, (3) inactivation of enzymatic activity by EDTA, (4) color code conversion using a complementary DNA probe, and (5) assembly of ssDNA–AuNPs assisted by brief centrifugation.

The assay starts with annealing the probe DNA (mLinker) with target DNA (mtKRAS or wtKRAS), followed by enzymatic cleavage using ExoIII. The enzymatic reaction amplifies the difference between mtKRAS and wtKRAS. After selective digestion with mLinker, EDTA is added to inactivate the enzyme. Subsequently, color code conversion is achieved by introducing a complementary DNA probe (c-mLinker). The final step involves the assembly of ssDNA–AuNPs with a brief centrifugation for visual detection.

Critical linker concentration, enzyme inactivation using EDTA, and colour conversion using c-mLinker were optimized. The assay demonstrated successful SNP detection with a visual readout. The introduction of c-mLinker enabled a “light-on" response for mtKRAS, making it visually distinct from wtKRAS and control samples.

The LOD of the assay was determined to be 67 pM, equivalent to 1% mutant KRAS in a ctDNA sample. The assay effectively detected mtKRAS in the presence of wtKRAS background, with visual differentiation at 1% mtKRAS. The unambiguous POC readout, with precipitates only for mtKRAS samples, makes it suitable for resource-limited settings in cancer diagnosis and prognosis.

Conclusion:

In summary, they have developed a rapid point-of-care (POC) method for visually detecting single nucleotide polymorphisms (SNPs) in circulating tumor DNA (ctDNA), specifically targeting a 151 nt KRAS sequence. This colorimetric assay combines enzymatic isothermal amplification (ExoIII-based cyclic cleavage) with centrifugal amplification, introducing a brief spinning step to induce distinct precipitation for mutant KRAS (mtKRAS) down to a limit of detection (LOD) of 67 pM. The assay operates effectively in a 99% background of normal ctDNA. Key design concepts, including defining a “critical linker concentration," room temperature enzyme inactivation using EDTA, and colour code conversion, enhance the assay’s robustness and clinical utility for fast turnaround times in POC settings. These improvements make the method promising for non-invasive cancer diagnosis and prognosis.

Reference:

WANG, Y., KONG, S.L. AND DI SU, X., 2020. A centrifugation-assisted visual detection of SNP in circulating tumor DNA using gold nanoparticles coupled with isothermal amplification. RSC advances, 10(3), pp.1476-1483.