Amplification-Free Strategy for miRNA Quantification in Human Serum Using Single Particle ICP–MS and Gold Nanoparticles as Labels

In the research article published in ACS Analytical Chemistry journal the researchers from the Department of Physical and Analytical Chemistry, and the Department of Functional Biology (Physiology), University of Oviedo, Spain presented novel analytical strategy for detecting miRNA in human plasma without relying on nucleic acid amplification.

MicroRNAs (miRNAs) are short RNA sequences that play a key role in gene expression and respond to physiological changes, such as those induced by physical exercise. This study presents a novel analytical strategy for detecting miRNA in human plasma without relying on nucleic acid amplification (eg PCR). The method uses a double hybridization process with a magnetic microparticle and a gold nanoparticle for highly selective detection, even at single nucleotide variations. The approach is validated against rt-qPCR and can differentiate between sedentary and active individuals based on miRNA levels. Additionally, this versatile platform can be easily adapted for other miRNA sequences.

The original article

Amplification-Free Strategy for miRNA Quantification in Human Serum Using Single Particle ICP–MS and Gold Nanoparticles as Labels

Sara González Morales, Carlos López-Portugués, Manuel Fernández-Sanjurjo, Eduardo Iglesias-Gutiérrez, María Montes Bayón, and Mario Corte-Rodríguez

Analytical Chemistry 2024 96 (30), 12414-12423

DOI: 10.1021/acs.analchem.4c01904

licensed under CC-BY 4.0

Abstract

MicroRNAs (miRNAs), which are short single-stranded RNA sequences between 18 and 24 nucleotides, are known to play a crucial role in gene expression. Changes in their expression are not only involved in many diseases but also as a response to physiological changes, such as physical exercise. In this work, a new analytical strategy for the sensitive and specific analysis of miRNA sequences in human plasma is presented. The developed strategy does not depend on any nucleic acid amplification process and can be obtained in direct correlation to the number of events obtained by using single-particle ICP–MS measurements. The high selectivity of the assay (up to single nucleotide polymorphisms) can be achieved by a double hybridization process of the target miRNA with a complementary capture oligonucleotide that is conjugated to a magnetic microparticle and simultaneously with a complementary reporter oligonucleotide conjugated to a gold nanoparticle. Thanks to the novel approach followed in this method, the stoichiometry of the oligonucleotide-nanoparticle conjugates does not need to be addressed for the quantification of the target miRNA, which also represents a big advantage over other similar methods. The optimized method is applied to the determination of a miRNA as a biomarker of physical exercise in non-spiked human serum samples, and the results are validated against rt-qPCR. The achieved sensitivity permits the direct differentiation among sedentary and sportive subjects. This general platform can be easily applied to any other sequence by only modifying the capture and reporter oligonucleotides, paving the way for multiple clinically interesting applications.

Introduction

Microribonucleic acids (miRNAs) are small noncoding RNA biomolecules with a length between 18 and 24 nucleotides that are involved in different cellular processes, such as gene expression modulation. In fact, the main function of miRNAs consists of their union with mRNAs by base complementarity, either preventing their translation or inducing their degradation, resulting in both cases in a negative post-transcriptional regulation of gene expression. Over 60% of protein-coding genes are known to be regulated by different miRNAs. (1)...

...In this work, miRNA quantification is based on the specificity of nucleic acid hybridization and the highly sensitive and specific nanoparticle counting capabilities of individual nanoparticles by SP-ICP-MS. The amplification-free workflow that we propose is based on the hybridization of a specific target miRNA sequence (miR-16-5p) with two DNA sequences that are each complementary to half of the target, respectively. One of them is conjugated to a magnetic microparticle (capture probe) that is used for the enrichment and washing of the target sequence. The other half-complementary sequence is conjugated to a gold nanoparticle that is detected by SP-ICP-MS (detection probe). Although the analytical strategy is comparable to this from Xu et al., (34) it is important to note that our methodology is applied to quantify different levels of expression of miRNA sequences as short as 22 nucleotides that are naturally present in human serum samples. Scheme 1 summarizes the main differences between the proposed strategy and other methods for miRNA quantification. Therefore, this methodology represents a step forward in facing challenges like miRNA pre-concentration in a complex matrix with the important role of quantification, which is crucial to extracting conclusions depending on the expression levels.

Anal. Chem. 2024, 96, 30, 12414-12423: Scheme 1. Main Differences of the Methodology Proposed in This Article in Comparison With Other Traditional Techniques.

Experimental Section

Instrumentation

The measurements were performed by using the iCAP TQ triple quadrupole ICP–MS system from Thermo Fisher Scientific. All measurements, including single particle ICP–MS measurements, were done using the standard configuration for sample introduction, including the MicroMist nebulizer at a sample flow rate of 0.4 mL min–1 and a cyclonic spray chamber because a high transport efficiency was not crucial for this application. The sample introduction system was operated in combination with an ASX-560 autosampler from Teledyne Cetac. The ICP–MS was tuned daily to maximize sensitivity while keeping the formation of oxides and doubly charged species below 3 and 5%, respectively.

For spectrophotometric measurements of nucleic acid concentrations, a NanoDrop spectrophotometer (Thermo Fisher Scientific) was used. A transmission electron microscope MET JEOL-JEM-2100F was used to obtain images of the nanoparticles. A scanning electron microscope MEB JEOL-6610LV with EDX microanalysis was used for the images and elemental analysis of microparticles. Other basic laboratory instrumentation was also used, including an analytical precision balance, an ultrasonic bath, a vortex mixer, and a block heater...

Results and Discussion

Characterization of Naked and Conjugated Gold Nanoparticles

The gold nanoparticles used as metal labels for the detection probe were characterized both before and after their conjugation with the DNA oligo by SP-ICP-MS, transmission electron microscopy (TEM), and DLS. The results of SP-ICP-MS, shown in Table 1 and Figure 1, reveal that the obtained mean diameters were 23.3 ± 3.3 nm, which was slightly larger than the value provided by the manufacturer (22 nm). When a Student-t test was performed to compare the mean diameters of the conjugated and original nanoparticles, no statistical differences between them or compared to the value given by the manufacturer was observed at a 95% confidence level. Under these conditions, the size limit of detection was calculated as 18 nm, well below the size of the nanoparticles. Both sets of particles showed a narrow distribution, as can be seen in Figure 1, although the nanoparticles showed a more monodisperse distribution after the conjugation (Figure 1B), probably due to an increased stability in solution when coated with negatively charged oligonucleotides...

Anal. Chem. 2024, 96, 30, 12414-12423: Table 1. Theoretical (Specified by Manufacturer) and Experimental Diameter for the Pegylated Gold Nanoparticles Before (Bare) and After (Conjugated) Conjugation With the Oligo_ᵃ.

Anal. Chem. 2024, 96, 30, 12414-12423: Figure 1. Histograms showing the diameters distribution of the gold nanoparticles analyzed by SP-ICP-MS. (A) (in black) Unconjugated nanoparticles and (B) (in red) nanoparticles after being conjugated with the oligonucleotide probe, with a shift to higher diameter values.

Conclusions

In this study, we introduce a novel amplification-free miRNA analysis method using magnetic microparticles for capture and preconcentration and gold nanoparticles as labels that amplify the response for ICP–MS detection. Eliminating the need for nucleic acid sequence amplification, our approach streamlines the analysis process while reducing potential sources of error that are common in conventional techniques. Moreover, it circumvents the often challenging considerations of oligonucleotide-nanoparticle conjugation stoichiometry. Importantly, this methodology also demonstrates remarkable resilience to batch-to-batch variations in conjugation processes, which are accounted for in daily calibration.
The high selectivity of the assay, thanks to the complementarity interactions between probes and analytes, has been demonstrated, achieving the possibility of clearly discriminating a 2-base mismatch. Even more, the response to single nucleotide polymorphisms is seriously affected, and these kinds of important sequence variations can also be also discriminated. The developed method, which has been validated against the gold-standard RT-qPCR, can be easily modified to detect any miRNA sequence by changing only the detection and capture probes. This allows for high versatility with minimal optimization for the detection of any miRNA sequence.